Systems tools and methods of assaying biological materials using spatially-addressable arrays

ABSTRACT

Systems, tools and methods of assaying biological material are used to perform complex sandwich hybridization assays. The tools used comprise biological solution probes that are customized for each assay. The solution probe comprises a first region for hybridizing to a probe, in a generic set of capture probes on a universal assay apparatus, and a second region for hybridizing to a target in a sample. The solution probe assembles the target to the assay apparatus by hybridizing the second region to the target and the first region to the capture probe. In array assays, one or more biological samples, having one or more targets per sample, can be multiplexed on the same universal array comprising the generic set of capture probes in an array pattern of features on the substrate. The customized solution probe addresses and assembles a predetermined target-sample combination onto the array at a corresponding capture probe address location. The systems, tools and methods have specificity and sensitivity by systematically providing a reduced likelihood of cross-hybridizations and intramolecular structures in the probes. Specificity and sensitivity of the assay are provided by the incorporation of a chemically modified monomer in the capture probe and a similarly modified monomer complement in the first region of the solution probe. The modified monomers preferentially hybridize with each other. When the probe and respective probe region are oligonucleotides, the complementary modified nucleotides have a reversed polarity relative to the polarity of the respective probe and probe region. The complementary reversed polarity nucleotides form a thermodynamically more stable hybridization to each other than a hybridization between the reversed polarity nucleotide and a complementary nucleotide whose polarity is not similarly reversed.

TECHNICAL FIELD

This invention relates to analytical tools and methods for monitoringlevels of gene expression and mutations in gene sequences. Inparticular, the invention relates to assay systems, tools and methodswith enhanced specificity and sensitivity that are capable ofmultiplexing one or more sample(s), having one or more targetmaterial(s) per sample, on a single array.

BACKGROUND ART

Conventional analysis of biological materials, such as DNA, RNA,proteins, antibodies, ligands and the like, employs a basichybridization assay which comprises a substrate or support havingbiological material chemically bound thereto. The biological materialmay be either biological “probes” of known molecular make-up or“targets” having an unknown characteristic to be determined. For thepurposes of simplicity, hereinafter the material bound to the substratewill be referred to as probes.

The probes are hybridized with a target sample and the hybridizationresults are analyzed. The hybridization results reveal information aboutthe targets based on what is known about the probes. The surface boundprobes are typically formed of DNA oligonucleotides, cDNA's, PCRproducts, proteins, antibodies, antigens, receptors, ligands, and thelike, that are complementary to the biological target material undertest.

Another conventional assay is the sandwich hybridization assay. Sandwichhybridization assays use probes designed with a sequence region that iscomplementary to the target under test and a separate sequence region ora separate binding partner that is complementary to a sequence, orspecific to a binding partner, on a support. The probes are hybridizedwith the target sample and with its complement on the support in a twostep process. Variations on this basic scheme have been developed toenhance accuracy, facilitate separation of duplexes and amplify signalsfor detection during analysis (see for example, U.S. Pat. Nos.4,868,105; 5,200,314; 5,635,352; and 5,681,697, issued to Urdea (orUrdea et al.) and U.S. Pat. Nos. 5,681,702 and 5,780,610, both issued toCollins et al.).

However, a drawback to the sandwich hybridization technique is crosshybridization. For example, if the target material hybridizes to thewrong region of the probe, then the probe does not hybridize with itsappropriate complement on the support. This may yield a false negativeresult. Conversely, if the target material hybridizes incorrectly to thesequence on the support, a false positive result may occur. Thus,information about the target material becomes less accurate. There hasbeen much effort in developing methods for minimizingcross-hybridization in sandwich hybridization assays. U.S. Pat. No.5,604,097, U.S. Pat. No. 5,635,400 and U.S. Pat. No. 5,846,719, issuedto S. Brenner and Brenner et al., respectively (hereinafter “Brenner”),disclose methods of sorting polynucleotides in basic hybridizationassays using‘minimally cross-hybridization’ sets of oligonucleotidetags. Brenner is silent on using the methods of sorting for sandwichhybridization assays. Oligonucleotide tags from the set of tags areattached to a sample of polynucleotides under test. The polynucleotideswith oligonucleotide tags attached are immobilized on a solid phasesupport by hybridizing the tags to a complementary sequence on thesupport.

Brenner discloses a general algorithm and computer program for computingminimally cross-hybridizing sets of tags and complements. Brenner's testfor “minimally cross-hybridizing” is based upon the conventionaltechnique of symbolic matching of sequences. Although useful in somecases, the conventional symbolic matching technique has drawbacks thataffect the technique's ability to effectively discriminate againstcross-hybridizations. Since some base mismatches are much lessdestabilizing to the duplex Tm than other mismatches, the method ofBrenner is capable of generating mismatch sequences which are actuallycapable of cross-hybridizing. The conventional method and the methoddisclosed by Brenner do not take in account cross-hybridizingmismatches. In addition, Brenner does not teach a method for protectingagainst the formation of intramolecular structures. These structures,such as hairpins, will inhibit the correct duplex formation between tagsand their complements. If cross-hybridizing mismatches andintramolecular structures were screenable according to Brenner's method,the number of tags and complement sets after such screening, which wouldactually qualify as “minimally cross-hybridizing”, would be greatlyreduced. With state-of-the-art arrays containing more than 10,000features, the tag sets disclosed by Brenner would have to have longerlengths than that disclosed by Brenner in order to yield a high enoughnumber to accommodate such an array. However, longer length tags andcomplements are more expensive to synthesize.

Thus, it would be advantageous to have a large number of ‘tag andcomplement’ sets, for example, for use in diagnostic assays ofbiological materials, wherein the tag and complement lengths are asshort as possible to save on cost. Further, it would be advantageous ifthe specificity between the tags and their complements was increased toavoid or minimize cross-hybridizations and still further if thesensitivity between the tags and their complements was increased bydecreasing the probability of intramolecular structures, such ashairpins, within the sequences. Still further, it would be advantageousif such tag and complement sets could be adapted to sandwichhybridization assays using arrays of over 10,000 features.

U.S. Pat. No. 5,399 676, U.S. Pat. No. 5,527,899, and U.S. Pat. No.5,721,218 issued to B. Froehler, disclose using oligonucleotides with“inverted polarity” for forming anti-sense probes having an extendedtriple helix with a double-helical nucleotide duplex. The anti-senseprobes are used in clinical intervention applications to decreasespecific RNA translation. Froehler discloses that the inverted polarityoligonucleotides can skip from one complementary strand in the duplex tothe other as its polarity shifts. Such inverted polarity also stabilizesthe single-stranded oligonucleotides to exonuclease degradation.However, Froehler is silent on using inverted polarity oligonucleotidesfor minimizing cross hybridization in diagnostic assays. In addition,Froehler discloses using probes that actually have specificintramolecular structures, which is consistent with the use ofanti-sense probes in clinical intervention applications.

Thus, it would be advantageous to have tools and methods fordiagnostically assaying one or more biological sample(s), having one ormore target(s) per sample, on a single array, using sandwichhybridization assay techniques. In addition, it would be advantageousfor the tools and methods to have increased specificity betweencomplementary probe sequences to minimize the likelihood ofcross-hybridization between biological materials in a systematicfashion. Moreover, it would be advantageous for such tools and methodsto have increased sensitivity between complementary probe sequences tominimize the likelihood of intramolecular structures within the probes.The increased specificity and sensitivity of such tools and methodswould increase the accuracy and usefulness of sandwich hybridizationassays, especially on an array.

SUMMARY OF THE INVENTION

The present invention provides sandwich hybridization assay systems,biological tools and methods of diagnostically assaying biologicalmaterials. The present invention is particularly useful for sandwichhybridization assays on arrays and provides an addressable,self-assembling array. The systems, tools and methods are capable ofmultiplexing one or more sample(s), having one or more target(s) persample on a single array. Moreover, the systems, tools and methods ofthe invention have good specificity by systematically providing areduced likelihood of cross-hybridizations from occurring, and goodsensitivity by systematically providing a reduced likelihood ofintramolecular structures from forming. The sandwich hybridization assaysystems, tools and methods of the present invention provide powerfulmeans for sorting, tracking, identifying, and determining othercharacteristics of biological target compounds for diagnosticapplications.

According to one aspect of the present invention, an assay system formultiplexing on a single array one or more biological sample(s), havingone or more biological target(s) per sample, is provided. The assaysystem for multiplexing comprises an array apparatus that has a firstplurality of biological probes, called capture probes, in an arraypattern of features on a substrate. Each capture probe in each featurelocation is different from the others in the first plurality. Eachdifferent capture probe is a different address on the array apparatus.

The assay system for multiplexing still further comprises a secondplurality of biological probes, called solution probes. Each solutionprobe comprises a first region and a second region. Each solution probeis different from others in the second plurality by comprising adifferent first region, and may comprise a different second region,depending on the assay. The first region of each solution probe iscomplementary to a respective capture probe on the array. The secondregion of the solution probe is complementary to a respective biologicaltarget in a sample. Thus, the solution probes essentially assemble ordeliver different biological target-and-biological sample combinationsbeing assayed on the array corresponding to the addresses of thedifferent first probes. The presence, quantity and/or other features ofparticular targets in particular samples are ascertainable depending onwhether the particular target-sample combinations have been assembled totheir respective capture probe location on the array during the assay.

According to another aspect of the present invention, an assay method ofmultiplexing on a single array one or more biological sample(s), havingone or more biological target(s) per sample, is provided. The assaymethod of multiplexing comprises the step of providing an arrayapparatus that has a first plurality of biological probes, calledcapture probes, in an array pattern of features on a substrate. Eachcapture probe of the first plurality is different and each differentcapture probe is located in a different feature location of the array.Each different capture probe is an address on the array apparatus.

The assay method of multiplexing further comprises the step of providinga second plurality of biological probes, called solution probes. Eachsolution probe of the second plurality has a first region and a secondregion. Each solution probe is different from other of the secondplurality by having a different first region. Each different firstregion is complementary to a different capture probe on the array. Thesecond region is complementary to a biological target from a biologicalsample, and therefore, the second region may be the same or different oneach solution probe of the second plurality, depending on the assay tobe performed. As mentioned above for the system, the solution probesessentially assemble or deliver the different biologicaltarget-and-biological sample combinations being assayed on the arraycorresponding to the addresses of the different capture probes. Theassay method of multiplexing still further comprises the steps ofassembling the biological target(s) from the sample(s) to the array, andremoving unassembled biological materials from the array and analyzingthe assay results using conventional methods. The presence of thebiological target(s) from respective biological sample(s) atcorresponding capture probe feature locations on the array indicates,among other things, whether and how much of particular biologicaltargets exist in particular biological samples.

During the step of assembling, a target from a sample will hybridizewith a complementary second region of a solution probe and the firstregion of this solution probe will hybridize with a complementarycapture probe on the array corresponding to the target-samplecombination. The solution probes of the system essentially assemble or“address” the targets to the array in a predetermined (pre-addressed)fashion during the assay. The assay system and method for multiplexingof the present invention advantageously provide an assay that isself-assembling and addressable, and capable of sorting for evaluationpurposes: one target from a plurality of different samples (orpatients), a plurality of different targets from one sample (patient),or a plurality of different targets from a plurality of differentsamples on the same array. The hybridizations may be performedsimultaneous or preferably, in a two step hybridization process.

According to still another aspect of the present invention, a set ofbiological probes used in a sandwich hybridization assay of a biologicaltarget on an array of biological features is provided. The set ofbiological probes comprises a plurality of individual solution probesthat each comprises a first probe region, called an anti-capture region,complementary to a biological feature on the array. Each individualsolution probe further comprises a second probe region that iscomplementary to the biological target being assayed. Each solutionprobe in a particular set is different by comprising a differentanti-capture region. The anti-target region on each different solutionprobe of a set may be the same or different depending on the type ofassay being performed. Moreover, there may be multiple copies of eachdifferent solution probe in the set. There is a different set ofbiological probes for each type of biological material being assayed(e.g., nucleic acids, proteins, sugars, etc.).

Thus, each solution probe of a particular set may comprise first regionsselected from oligonucleotides, antibodies, antigens, ligands andreceptors, for example, depending on the biological make-up of thebiological features on the array. Further, the second regions of thisparticular set may comprise second regions selected from cDNA, PCRproducts, oligonucleotides, antibodies, antigens, ligands and receptors,for example, depending on the biological make-up of the biologicaltargets to be assayed. For example, each solution probe of a particularset may comprise a different antigen linked to the same or a differentcDNA, wherein the different antigens are complementary to a plurality ofdifferent antibody features on an array and the cDNA is complementary tooligonucleotide (e.g., mRNA) target material(s) to be assayed. Thus,each set of solution probes is customized to a particular assay. Thegeneric set of capture probes on the universal array apparatus provides“addresses” corresponding to the location of the capture probes on thearray where target material being assayed is to be delivered and the setof solution probes essentially delivers the target material to itsrespective address during the assay. The set of biological probes areparticularly useful for multiplexing assays of one or more biologicalsample(s), having one or more biological target(s) per sample, on asingle array.

In still another aspect of the present invention, a system for assayingbiological materials having specificity and sensitivity is provided. Thesystem comprises an apparatus that has a first set of biologicalmaterial probes, called capture probes, on a substrate. Each captureprobe of the set comprises a sequence of monomers. Each set of captureprobes is generic to the biological material to be tested. Each captureprobe in the set may be different by having a different sequence ofmonomers. There may be multiple copies of each different capture probein the set.

The system having specificity and sensitivity further comprises a secondset of biological material probes, called solution probes. There is adifferent set of solution probes for each type of biological materialbeing tested. Each solution probe of the set comprises a first sequenceregion, called an anti-capture sequence, that is complementary to themonomers in the capture probe sequence for hybridizing or binding to thecapture probe, and a second region, called an anti-target region, forhybridizing or binding to the target. Each solution probe in aparticular set may be different by comprising a different anti-capturesequence. Further, each solution probe may comprise the same ordifferent anti-target region depending on the biological materials to beassayed. There may be multiple copies of each different solution probein the set. The set of solution probes assembles the target material tobe tested to the assay substrate by hybridizing the second region to thetarget and hybridizing the first region to the capture probes on thesubstrate. The hybridizations may be performed simultaneously orpreferably, in two hybridization steps.

In accordance with this aspect of the invention, the capture probes andthe anti-capture sequences on the solution probes each comprise acomplementary chemically modified monomer that preferentially hybridizeor bind to each other instead of to a complementary unmodified (or notsimilarly modified) monomer. The preference of the modified monomers ofthe present invention provides specificity and sensitivity to thesystem. The system is specific because the chemically modified monomerin the anti-capture sequences will preferentially hybridize or bind withthe complementary similarly modified monomer in the respective captureprobe, and likewise the modified monomer in the capture probe willpreferentially hybridize or bind with the similarly modified monomer inthe complementary anti-capture sequence. Thus, cross-hybridizations(i.e., hybridizations with mismatches) are less likely to occur. Thesystem is sensitive because the chemically modified monomers in thecapture probes and anti-capture sequences are less likely to hybridizeto complementary unmodified (or not similarly modified) monomers or tononcomplementary monomers present in the capture probes, solution probesor target sequences. Thus, hybridizations that cause intramolecularstructures within the capture probes or within the anti-capturesequences are less likely to occur.

In yet another aspect of the present invention, a method of assayingbiological materials having specificity and sensitivity is provided. Themethod comprises the step of providing an apparatus having a first setof biological probes, called capture probes, comprising a sequence ofmonomers, on a substrate.

The method having specificity and sensitivity further comprises the stepof providing a second set of biological probes, called solution probes.There is a different set of solution probes for each type of biologicalmaterial to be tested. Each solution probe of the set comprises a firstsequence region of monomers, called an anti-capture sequence region, anda second region, called an anti-target region. The anti-capture sequenceregion of each solution probe is complementary to the monomer sequenceof a capture probe in the set, and the anti-target region on thesolution probe is complementary to a biological target to be assayed.

The complementary capture probes and anti-capture monomer sequences eachinclude a complementary chemically modified monomer that prefers tohybridize or bind to each other instead of to a complementary monomerthat is not similarly modified.

The method of assaying biological materials having specificity andsensitivity further comprises the step of assembling the target materialto the substrate for evaluation. During the step of assembling, the setof solution probes is incubated with a target biological material tocause hybridization or binding between targets and respectivecomplementary anti-target regions of the solution probes. Further, theset of solution probes is incubated with the set of capture probes onthe assay apparatus to cause hybridization or binding betweencomplementary capture sequences and anti-capture sequences of thesolution probes. It is during the step of assembling that thecomplementary chemically modified monomers on the capture probes andanti-capture sequences preferentially hybridize to each other. Thehybridizations may be performed simultaneously or preferably, in twohybridization steps. After hybridization, the incubated substrate iswashed to remove unhybridized/unbound material and the results of theassay are analyzed, according to conventional methods.

In a preferred embodiment, the capture probe and anti-capture sequencesare oligonucleotides and the chemically modified monomers in the captureprobes and anti-capture sequences are reversed polarity nucleotidesrelative to the polarity of the nucleotides of the respective sequence.The anti-capture sequence, having a nucleotide with reversed polarity,is complementary to the nucleotide with reversed polarity on the captureprobe. The complementary reversed polarity nucleotides prefer tohybridize to each other because they form a thermodynamically morestable hybridization than a hybridization between a reversed polaritynucleotide and its complementary nucleotide whose polarity is notsimilarly reversed.

The chemical modification introduced into the probes of the system andmethod essentially improves the likelihood that the appropriate orintended capture probes and solution probes will specifically andsensitively hybridize or bind together. Advantageously, the system andmethod of assaying essentially systematically provide a reducedlikelihood of cross-hybridizations between the capture probes andsequences that are not complementary to the capture probes, such asnon-complementary anti-capture sequences of the solution probes,anti-target regions of solution probes, or target sequences. Further,the system and method having specificity and sensitivity essentiallysystematically provide a reduced likelihood of cross-hybridizationbetween anti-capture sequences of the solution probes and sequences thatare not complementary to the anti-capture sequences, such asnon-complementary capture sequences on the array, other anti-capturesequences, anti-target sequences of solution probes, or targetsequences. In addition, the system and method having specificity andsensitivity essentially systematically provide a reduced likelihood ofany undesirable formation of intramolecular structures within thecapture probes and anti-capture sequences of the invention. The systemand method have a reduced likelihood of cross-hybridizations andintramolecular structures because the probes comprise modified monomersthat prefer to hybridize to each other rather than to a complementaryunmodified (or not similarly modified) monomer.

The system and method having specificity and sensitivity are useful insandwich hybridization assays, sandwich hybridization assays usingarrays, and in particular, in the system and method described above formultiplexing one or more biological sample(s), having one or moretarget(s) per sample on a single array. When used in multiplexing arrayassay applications, the system and method having specificity andsensitivity advantageously provide assays with specific and sensitiveaddressing and self-assembling capabilities.

The system and method having specificity and sensitivity of theinvention uses both chemically modified monomers and monomers that arenot modified (or not modified in a similar fashion to the chemicallymodified monomers of the invention), in the same capture probes andanti-capture sequences. Using both chemically modified monomers and notsimilarly modified monomers increases the number of different monomers(“letters”) available with which to make new sequences (“words”) for thecapture probes and anti-capture sequences of the invention. Thus, alarger number of different capture probe sequences and theircomplementary anti-capture sequences are readily and systematicallygenerated by the present invention that are specific and sensitivecompared to conventional assays.

In the preferred embodiment of oligonucleotide capture probes andanti-capture sequence regions of the solution probes of the system andmethod having specificity and sensitivity, there are at least eightnucleotides (four with one polarity and four counterparts withrespectively reversed polarity) from which to form the complementarycapture probe and anti-capture region sequences. Therefore, a muchlarger set of probes and anti-capture sequences with specificity andsensitivity are provided. Moreover, the specificity that thecomplementary reversed polarity nucleotides have for each other allowsthe length of the capture probes and anti-capture regions to be shorterthan conventional probes to save on cost.

The system and method having specificity and sensitivity advantageouslyprovide a set of greater than 10,000 probes, which are unique withrespect to each other. Mammalian genomes are estimated to contain over10,000 expressed genes. With multiple splicing variants, the number ofprobes needed to sample the expressed genes is even higher. Thus, if onewanted to assay for all of the expressed genes and variants on the samearray, one would need in excess of 10,000 biological capture features onthe array. The system and method having specificity and sensitivityprovide this capability.

In another aspect of the present invention, a kit is provided thatcomprises the assay apparatus of capture probes and the set of solutionprobes packaged with written instructions for use in accordance with oneor more assay methods of the present invention. The kit can be providedto users, such as diagnostic, research and/or analytical laboratories.The kit can comprise either (i) the array apparatus of capture probesand set of solution probes for multiplexing sandwich assays on arrays,or (ii) the apparatus of capture probes and a set of solution probes forsandwich assays having specificity and sensitivity, or (iii) acombination of both, comprising the array apparatus of capture probesand the set of solution probes having specificity and sensitivity formultiplexing sandwich assays, in accordance with one or more embodimentsof the invention.

Moreover, the systems, tools and methods of the present invention alsoprovide cost effective custom assays. Customization of an assay residesin the preparation of the second set of probes (solution probes), ratherthan in the preparation of the first set of probes (capture probes) orthe array apparatus of capture probes. Therefore, large numbers ofgeneric or universal assay substrates with bound capture probes can bemanufactured at a time, thereby saving in cost and turnaround time forcustom orders. This is particular advantageous for large numbers ofgeneric or universal array substrates of the invention. The second setof probes are prepared separately, as needed, or prepared in advance andstored separately in solution or dry, preferably frozen, until needed.The solution probes are customized to the biological materials of thegeneric or universal array apparatus and to the biological targets to beassayed.

BRIEF DESCRIPTION OF THE DRAWINGS

The various features and advantages of the present invention may be morereadily understood with reference to the following detailed descriptiontaken in conjunction with the accompanying drawings, where likereference numerals designate like structural elements, and in which:

FIG. 1 is a perspective view of one embodiment of an apparatus of thepresent invention illustrating an array of capture probe featurelocations on a substrate.

FIG. 2 is a magnified side view illustrating biological targetsassembled to one embodiment of the system of the present invention afteran assay.

FIG. 3 is a block diagram illustrating a method of multiplexingbiological materials on an array in accordance with one embodiment ofthe invention.

FIG. 4A is a diagram illustrating multiplexing of a single target typefrom multiple samples on a single array in accordance with oneembodiment of the system of the present invention.

FIG. 4B is a diagram illustrating multiplexing of multiple differenttargets from one sample on a single array using the system according toone embodiment of the present invention.

FIG. 4C is a diagram illustrating multiplexing of multiple differenttargets from multiple different samples on a single array using thesystem according to one embodiment of the present invention.

FIG. 5 is a magnified view illustrating the preferred embodiment of thesystem having specificity and sensitivity of the present invention afteran assay.

FIG. 6 is a diagram illustrating another embodiment of the system havingspecificity and sensitivity of the present invention comprising across-linking monomer pair.

FIG. 7 is a block diagram illustrating the method of assaying havingspecificity and sensitivity in accordance with one embodiment of theinvention.

FIG. 8A is a diagram of the system having specificity and sensitivityafter an assay illustrating a correct hybridization between respectiveprobes and sequence regions of the invention and a target sample.

FIGS. 8B, 8C and 8D are diagrams of the system for multiplexing after anassay illustrating possible cross-hybridizations and intramolecularstructures.

MODES FOR CARRYING OUT THE INVENTION

Definitions

The following terms are intended to have the following general meaningsas they are used herein:

Polynucleotide—a compound or composition that is a polymeric nucleotideor nucleic acid polymer. The polynucleotide may be a natural compound ora synthetic compound. In the context of an assay, the polynucleotide canhave from about 5 to 5,000,000 or more nucleotides. The largerpolynucleotides are generally found in the natural state. In an isolatedstate the polynucleotide can have about 30 to 50,000 or morenucleotides, usually about 100 to 20,000 nucleotides, more frequently500 to 10,000 nucleotides. It is thus obvious that isolation of apolynucleotide from the natural state often results in fragmentation.The polynucleotides include nucleic acids, and fragments thereof, fromany source in purified or unpurified form including DNA, double-strandedor single stranded (dsDNA and ssDNA), and RNA, including t-RNA, m-RNA,r-RNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA/RNAhybrids, or mixtures thereof, genes, chromosomes, plasmids, the genomesof biological materials such as microorganisms, e.g. bacteria, yeasts,viruses, viroids, molds, fungi, plants, animals, humans, and the like.The polynucleotide can be only a minor fraction of a complex mixturesuch as a biological sample. Also included are genes, such as hemoglobingene for sickle-cell anemia, cystic fibrosis gene, onocogenes, cDNA, andthe like.

Polynucleotides include analogs of naturally occurring polynucleotidesin which one or more nucleotides are modified over naturally occurringnucleotides. Polynucleotides then, include compounds producedsynthetically (for example, PNA as described in U.S. Pat. No. 5,948,902and the references cited therein, all of which are incorporated hereinby reference) which can hybridize in a sequence specific manneranalogous to that of two naturally occurring polynucleotides.

The polynucleotide can be obtained from various biological materials byprocedures well known in the art. The polynucleotide, where appropriate,may be cleaved to obtain a fragment that contains a target nucleotidesequence, for example, by shearing or by treatment with a restrictionendonuclease or other site specific chemical cleavage method, such aslimited RNase digestion, to produce smaller RNA fragments.

For purposes of this invention, the polynucleotide, or a cleavedfragment obtained from the polynucleotide, will usually be at leastpartially denatured or single stranded or treated to render it denaturedor single stranded. Such treatments are well known in the art andinclude, for instance, heat or alkali treatment, or enzymatic digestionof one strand. For example, double stranded DNA (dsDNA) can be heated at90-100° C. for a period of about 1 to 10 minutes to produce denaturedmaterial, while RNA produced via transcription from a dsDNA template isalready single stranded.

Oligonucleotide—a polynucleotide, usually single stranded, usually asynthetic polynucleotide but may be a naturally occurringpolynucleotide. The oligonucleotide(s) are usually comprised of asequence of at least 5 nucleotides, usually, 10 to 100 nucleotides, moreusually, 20 to 50 nucleotides, preferably, 10 to 30 nucleotides, morepreferably, 20 to 30 nucleotides, and desirably about 25 nucleotides inlength.

Various techniques can be employed for preparing an oligonucleotide.Such oligonucleotides can be obtained by biological synthesis or bychemical synthesis. For short sequences (up to about 100 nucleotides),chemical synthesis will frequently be more economical as compared to thebiological synthesis. In addition to economy, chemical synthesisprovides a convenient way of incorporating low molecular weightcompounds and/or modified bases during specific synthesis steps.Furthermore, chemical synthesis is very flexible in the choice of lengthand region of target polynucleotides binding sequence. Theoligonucleotide can be synthesized by standard methods such as thoseused in commercial automated nucleic acid synthesizers. Chemicalsynthesis of DNA on a suitably modified glass or resin can result in DNAcovalently attached to the surface. This may offer advantages in washingand sample handling. For longer sequences standard replication methodsemployed in molecular biology can be used such as the use of M13 forsingle stranded DNA as described in J. Messing (1983) Methods Enzymol.101:20-78.

In situ synthesis of oligonucleotide or polynucleotide probes on thesubstrate is performed in accordance with well-known chemical processes,including, but not limited to sequential addition of nucleotidephosphoramidites to surface-linked hydroxyl groups, as described by T.Brown and Dorcas J. S. Brown in Oligonucleotides and Analogues APractical Approach, F. Eckstein, editor, Oxford University Press,Oxford, pp. 1-24 (1991), and incorporated herein by reference. Indirectsynthesis may be performed in accordance biosynthetic techniques (e.g.polymerase chain reaction “PCR”), as described in Sambrook, J. et al.,“Molecular Cloning, A Laboratory Manual”, 2^(nd) edition 1989,incorporated herein by this reference.

Other methods of oligonucleotide synthesis include, but are not limitedto solid-phase oligonucleotide synthesis according to thephosphotriester and phosphodiester methods (Narang, et al., (1979) Meth.Enzymol. 68:90), and to the H-phosphonate method (Garegg, P. J., et al.,(1985) “Formation of internucleotidic bonds via phosphonateintermediates”, Chem. Scripta 25, 280-282; and Froehler, B. C., et al.,(1986a) “Synthesis of DNA via deoxynucleoside H-phosphonateintermediates”, Nucleic Acid Res., 14, 5399-5407, among others) andsynthesis on a support (Beaucage, et al. (1981) Tetrahedron Letters22:1859-1862) as well as phosphoramidate techniques (Caruthers, M. H.,et al., “Methods in Enzymology,” Vol. 154, pp. 287-314 (1988) and othersdescribed in “Synthesis and Applications of DNA and RNA,” S. A. Narang,editor, Academic Press, New York, 1987, and the references containedtherein, and nonphosphoramidite techniques. The chemical synthesis via aphotolithographic method of spatially addressable arrays ofoligonucleotides bound to glass surfaces is described by A. C. Pease, etal., Proc. Nat. Aca. Sci. USA (1994) 91:5022-5026. Oligoribonucleotidesynthesis using phage RNA polymerase and ribonucleoside triphosphates isdescribed by Milligan, J. F., et al., (1987) “Oligoribonucleotidesynthesis using T7 RNA polymerase and synthetic DNA templates”, Nucl.Acids Res. 15, 8783-8798; and using protected ribonucleosidephosphoramidites and chemical synthesis is described by Wu T., et al.,(1989) “Prevention of chain cleavage in the chemical synthesis of2′-O-silylated oligoribonucleotides”, Nucl. Acids Res. 17, 3501-3517,among others.

For the purposes of the invention, the term “oligonucleotide” includesthe term “polynucleotide”, unless stated otherwise.

Oligonucleotide probe—an oligonucleotide employed to bind to anotheroligonucleotide.

Monomer—A member of the set of small molecules which can be joinedtogether to form a polymer. The set of monomers includes but is notrestricted to, for example, the set of common L-amino acids, the set ofD-amino acids, the set of synthetic amino acids, the set of nucleotidesand modified nucleotides, and the set of pentoses and hexoses. Otherexamples include abasic phosphodiesters, such as polyethers, andprotein-nucleic acid (PNA) hybrids. As used herein, monomers refers toany member of a basis set for synthesis of a polymer. The monomer may benatural or synthetic. For example, dimers of the 20 naturally occurringL-amino acids form a basis set of 400 monomers for the synthesis ofpolypeptides. Different monomers may be used at successive steps in thesynthesis of a polymer. Furthermore, a monomer may include protectedmembers that are modified after synthesis.

Modified monomer—a naturally occurring monomer, obtained from a naturalsource or produced synthetically, that is chemically modified to add,replace, substitute, delete, or otherwise change one or more groups orbonds contained in the monomer. For the purposes of the invention, amonomer is modified, as mentioned above, to cause the modified monomerto preferentially hybridize or bind to another complementary monomerthat is similarly modified.

Nucleotide—the monomeric unit of nucleic acid polymers, i.e., DNA andRNA, whether obtained from a natural source or produced synthetically,which comprises a nitrogenous heterocyclic base, which is a derivativeof either a purine or pyrimidine, a pentose sugar, and a phosphate (orphosphoric acid). When the phosphate is removed, the monomeric unit thatremains is a “nucleoside”. Thus a nucleotide is a 5′-phosphate of thecorresponding nucleoside. When the nitrogenous base is removed from thenucleotide, the monomeric unit that remains is a “phosphodiester”. Forthe purposes of the invention, “nucleotide” includes its correspondingnucleoside and phosphodiester, and “oligonucleotide” includes itscorresponding oligonucleoside and oligophosphodiester, unless indicatedotherwise.

Modified nucleotide—a modified monomer in a nucleic acid polymer thatcontains a modified base, sugar and/or phosphate group. The modifiednucleotide can be naturally occurring or produced by a chemicalmodification of a nucleotide either as part of the nucleic acid polymeror prior to the incorporation of the modified nucleotide into thenucleic acid polymer. For example, the methods mentioned above for thesynthesis of an oligonucleotide may be employed. In another approach amodified nucleotide can be produced by incorporating a modifiednucleoside triphosphate into the polymer chain during an amplificationreaction. Examples of modified nucleotides, by way of illustration andnot limitation, include dideoxynucleotides, derivatives or analogs thatare biotinylated, amine modified, alkylated, fluorophore-labeled, andthe like and also include phosphorothioate, phosphite, ring atommodified derivatives, and so forth. The present invention is directed,in part, to a particular type of chemical modification to one or morenucleotides.

Hybridization (hybridizing) and binding—to associate together. In thecontext of nucleotide sequences these terms are used interchangeablyherein. The ability of two nucleotide sequences to hybridize with eachother is based on the degree of complementarity of the two nucleotidesequences, which in turn is based on the fraction of matchedcomplementary nucleotide pairs. The more nucleotides in a given sequencethat are complementary to another sequence, the more stringent theconditions can be for hybridization and the more specific will be thebinding of the two sequences. Increased stringency is achieved byelevating the temperature, increasing the ratio of co-solvents, loweringthe salt concentration, and the like. In the context of ligand/receptor,antibody/antigen, etc., binding depends on the affinity each of thespecific binding pair for the other and means a relatively stable bondbetween respective pairs.

In accordance with the invention, the conventional hybridizationsolutions and processes for hybridization can be used, such as thosedescribed in J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, Ed. 2^(nd), 1989, vol. 1-3, incorporated herein by reference.Conditions for hybridization typically include (1) high ionic strengthsolution, (2) at a controlled temperature, and (3) in the presence ofcarrier DNA and detergents and divalent cation chelators, all of whichare well known in the art.

Complementary—A term directed to the affinity that one biologicalmaterial has for binding to another biological material, such as membersof a specific binding pair, as defined below. For example, particularantibodies are complementary to particular antigens, particularreceptors to ligands and particular nucleotide to other nucleotides.With respect to nucleotide complements, two sequences are complementarywhen the sequence of one can bind to the sequence of the other in ananti-parallel sense wherein the 3′-end of each sequence binds to the5′-end of the other sequence and each A, T(U), G, and C of one sequenceis then aligned with a T(U), A, C, and G, respectively, of the othersequence. Non-standard base pairing is also possible with nucleotidecomplements, for instance, the sequences may be parallel to each otherand non-Watson-Crick base pairing may occur. Examples of the latter arecomplementary G=U or U=G base pairs in RNA sequences or complementaryG=T or T=G base pairs in DNA sequences.

Substrate or surface—a porous or non-porous water insoluble material.The surface can have any one of a number of shapes, such as strip,plate, disk, rod, particle, including bead, and the like. The substratecan be hydrophobic or hydrophilic or capable of being renderedhydrophobic or hydrophilic and includes inorganic powders such assilica, magnesium sulfate, and alumina; natural polymeric materials,particularly cellulosic materials and materials derived from cellulose,such as fiber containing papers, e.g., filter paper, chromatographicpaper, etc.; synthetic or modified naturally occurring polymers, such asnitrocellulose, cellulose acetate, poly (vinyl chloride),polyacrylamide, cross linked dextran, agarose, polyacrylate,polyethylene, polypropylene, poly (4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), etc.; either used by themselves or in conjunction with othermaterials; glass available as Bioglass, ceramics, metals, and the like.Natural or synthetic assemblies such as liposomes, phospholipidvesicles, and cells can also be employed. Common substrates used forarrays are surface-derivatized glass or silica, or polymer membranesurfaces, as described in Z. Guo et al. (cited above) and U. Maskos, E.M. Southern, Nucleic Acids Res 20, 1679-84 (1992) and E. M. Southern etal., Nucleic Acids Res 22, 1368-73 (1994), both incorporated herein byreference.

Immobilization of oligonucleotides on a substrate or surface may beaccomplished by well-known techniques, commonly available in theliterature. See, for example, A. C. Pease, et al., Proc. Nat. Acad. Sci.USA, 91:5022-5026 (1994); Z. Guo, R. A. Guilfoyle, A. J. Thiel, R. Wang,L. M. Smith, Nucleic Acids Res 22, 5456-65 (1994); and M. Schena, D.Shalon, R. W. Davis, P. O. Brown, Science, 270, 467-70 (1995), eachincorporated herein by reference.

Label—a member of a signal producing system. Usually the label is partof a target nucleotide sequence or an oligonucleotide probe, eitherbeing conjugated thereto or otherwise bound thereto or associatedtherewith. The label is capable of being detected directly orindirectly. Labels include (i) reporter molecules that can be detecteddirectly by virtue of generating a signal, (ii) specific binding pairmembers that may be detected indirectly by subsequent binding to acognate that contains a reporter molecule, (iii) oligonucleotide primersthat can provide a template for amplification or ligation or (iv) aspecific polynucleotide sequence or recognition sequence that can act asa ligand such as for a repressor protein, wherein in the latter twoinstances the oligonucleotide primer or repressor protein will have, orbe capable of having, a reporter molecule. In general, any reportermolecule that is detectable can be used.

The reporter molecule can be isotopic or nonisotopic, usuallynonisotopic, and can be a catalyst, such as an enzyme, a polynucleotidecoding for a catalyst, promoter, dye, fluorescent molecule,chemiluminescer, coenzyme, enzyme substrate, radioactive group, a smallorganic molecule, amplifiable polynucleotide sequence, a particle suchas latex or carbon particle, metal sol, crystallite, liposome, cell,etc., which may or may not be further labeled with a dye, catalyst orother detectable group, and the like. The reporter molecule can be afluorescent group such as fluorescein, a chemiluminescent group such asluminol, a terbium chelator such as N-(hydroxyethyl)ethylenediaminetriacetic acid that is capable of detection by delayedfluorescence, and the like.

The label can generate a detectable signal either alone or together withother members of the signal producing system. As mentioned above, areporter molecule can be bound directly to a nucleotide sequence or canbecome bound thereto by being bound to an specific binding pair (sbp)member complementary to an sbp member that is bound to a nucleotidesequence. Examples of particular labels or reporter molecules and theirdetection can be found in U.S. Pat. No. 5,508,178, the relevantdisclosure of which is incorporated herein by reference. When a reportermolecule is not conjugated to a nucleotide sequence, the reportermolecule may be bound to an sbp member complementary to an sbp memberthat is bound to or part of a nucleotide sequence.

Signal Producing System—the signal producing system may have one or morecomponents, at least one component being the label. The signal producingsystem generates a signal that typically relates to the presence oramount of a target polynucleotide in a medium. A signal producing systemmay be incorporated on the oligonucleotide probes and relates to thepresence of probes in a medium. The signal producing system includes allof the reagents required to produce a measurable signal. Othercomponents of the signal producing system may be included in thedeveloper solution and can include substrates, enhancers, activators,chemiluminescent compounds, cofactors, inhibitors, scavengers, metalions, specific binding substances required for binding of signalgenerating substances, and the like. Other components of the signalproducing system may be coenzymes, substances that react with enzymicproducts, other enzymes and catalysts, and the like. The signalproducing system provides a signal detectable by external means, by useof electromagnetic radiation, desirably by optical examination.Signal-producing systems that may be employed in the present inventionare those described more fully in U.S. Pat. No. 5,508,178, the relevantdisclosure of which is incorporated herein by reference.

Member of a specific binding pair (“sbp member”)—one of two differentmolecules, having an area on the surface or in a cavity thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as cognates or asligand and receptor (anti-ligand). These may be members of animmunological pair such as antigen-antibody, or may beoperator-repressor, nuclease-nucleotide, biotin-avidin, hormones-hormonereceptors, nucleic acid duplexes, IgG-protein A, DNA-DNA, DNA-RNA, andthe like, and for the invention, may include members of a cross-linkingpair.

Biolozical material—nucleic acids, such as DNA, RNA, polynucleotides,oligonucleotides, oligonucleotide probes, proteins, amino acids,antibodies, antigens, enzymes, coenzymes, ligands, receptors, hormonesand labels, and monomers thereof, and genes that specify any of theabove, and any other materials from any form of life and the syntheticversions of any of the above.

“Probe” or “Biological probe” means a biological material, such as amember of a specific binding pair of generally known make-up orcomposition, which is used to bind its complementary member of therespective specific binding pair to obtain information about a targetmaterial attached to the complementary member. The probe may becomprised of an oligonucleotide, antibody, antigen, ligand or areceptor, for example.

“Target”, “Target sample”, “Target material” or “biological target”means the biological material, synthetic or natural, which is under testor to be assayed. The target may be a oligonucleotide, or portionthereof, complementary to the oligonucleotide probe; a complementaryantigen, or portion thereof, to an antibody probe; a complementaryantibody, or portion thereof, to an antigen probe; a complementaryreceptor or ligand, or portion thereof, to a respective ligand orreceptor probe, for example.

“Sample” or “biological sample”—means a portion of a biologicalmaterial, either natural or synthetic, comprising one or more targetmaterials. A sample may be blood, urine, tissue, etc., or a componentthereof, for example, from a patient, either mammal, animal, bacterial,or viral, or any other form of life.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes systems, tools and methods used to assaybiological materials for diagnostic applications.

One or more biological samples having one or more biological targets persample are multiplexed on a single array using the assay system 10 ofthe present invention. Referring to FIG. 1, assay system 10 comprises anarray apparatus 11 having a substrate 12 with a surface 13 and a firstplurality of biological probes, hereinafter called the capture probes C,which is covalently bound or linked to the surface 13 of the substrate12 in an array pattern of features 14. The substrate can be made ofconventional materials defined above and can be any shape that supportsan array of features. Each capture probe C_(i), where i=1→n, of thefirst plurality may be different and each different capture probe C_(i)is located in a different feature location 14 _(i) on the array 11. In apreferred embodiment, there are multiple copies (e.g., >10,000molecules/um²) of each different capture probe C_(i) covalently bound toeach respective feature location 14 _(i). Each capture probe C_(i) willnon-covalently “capture” a target of interest during an assay. The firstplurality of capture probes C and therefore, the array apparatus 11, areuniversal or generic to the assay and to the biological materials beingassayed.

Not all of the capture probe features C are illustrated in FIG. 1 forsimplicity. In accordance with the invention, there may be over 10,000(n=1 to >10⁴) capture probe features C_(i) on a single array apparatus11, rendering the array system 10 capable of multiplexing as many uniquecombinations of biological samples-and-targets. Such an array system 10will support the analysis of genes from the human genome, for example.Referring to FIG. 1, each individual feature 14 _(i), for example, onthe array has a plurality of the same capture probes C_(i),respectively, covalently bound to the respective feature location.Capture probe C_(n) is the “nth” capture probe feature on the array thathas “n” total feature locations.

The capture probes C can either be synthesized in situ directly onto thesubstrate surface 13 or pre-synthesized and added as an intact specieswhich is then covalently linked to the surface 13 of the substrate 12using well-known conventional processes and materials. It is not theintent of the inventor to be limited to any synthesis method for theinvention. Any synthesis method will work and all methods are within thescope of the invention. Information about the compositional makeup ofeach capture probe C_(i) and the location on the array of each captureprobe C_(i) is maintained in a database. As a result, the array 11 isuniversal and can be manufactured in bulk quantities to reduce cost andturn around time. Likewise, the capture probes C for an assay aregeneric to the target material being assayed and can be manufactured inbulk quantities and added to the array substrate 12 as needed to reducecost and turn around time. Customization of an assay of the presentinvention is provided by the solution probes S, described below.

The assay system 10 further comprises a second plurality of biologicalprobes, namely the solution probes S. Each solution probe S_(i,j), wherej=1 to m, comprises a first region, called the anti-capture region,αC_(i), which is complementary and binds to a respective capture probeC_(i) on the array substrate 12. For example, for capture probe C₁, thecomplementary anti-capture region of the solution probe S_(1,j) is αC₁(and for capture probe C₂, the solution probe is S_(2,j) and thecomplementary anti-capture region is αC₂; for C₃, is S_(3,j) and αC₃; .. . for C_(n), is S_(n,m) and αC_(n), respectively).

Each solution probe S_(i,j) further comprises a second region, calledthe anti-target region, αT_(j), which is complementary and binds to atarget T_(j,k) of interest from a sample, or patient P_(k), where mequals the number of different targets in a sample P and k=1→x, where xequals the number of samples. The anti-capture region αC and theanti-target region αT of the solution probe S may be separatelymanufactured. The anti-target region αT may be linked to theanti-capture region αC of the solution probe S with a strongnon-covalent bond, and preferably, the two regions of the solution probeS are linked covalently. Alternatively, the anti-capture region αC andthe anti-target region αT may be regions of a continuous sequence ofmonomers of the same biological type that make up the solution probe S,wherein the linkage between the two regions is a conventional linkagebetween two monomers of a polymer.

The anti-capture regions αC of the set of the solution probes S aresimply the complements to the generic set of capture probes C on theuniversal array apparatus 11. Therefore, the anti-capture regions αC canbe manufactured separately and stored in bulk until needed to form acomplete solution probe S for an assay. Moreover, the anti-targetregions αT can be either synthesized directly on the anti-captureregions αC, or synthesized separately and linked to the anti-captureregions αC as an intact species using well known methods of synthesizingand linking. This provides a time and cost savings benefit.Alternatively, the anti-capture regions αC and the anti-target regionsαT of the solution probe S can be manufactured together when theinformation about samples P and targets T for the assay are known. Inaccordance with the invention, each capture probe C_(i) on the apparatus11 is an address and, for a given experiment, corresponds to apredetermined biological target from a biological sample (ortarget-sample combination T_(j)-P_(k)). The customization of the arrayis accomplished by the plurality of solution probes, which arecustomized to deliver the target-sample combinations to thecorresponding capture probe locations (i.e. “addresses”) on the arrayapparatus 11. In the preferred embodiment, these addresses are stored ina computer for the particular multiplexing assay.

To address a particular target T_(j)-sample P_(k) combination(hereinafter abbreviated as T_(j)-P_(k) or T_(j,k)) to a particularcapture probe C_(i) location 14 _(i) on the array apparatus 11 for anassay in accordance with the invention, the anti-capture region αC_(i)of a respective solution probe S_(i,j) must be complementary to thecapture probe C_(i) that corresponds to the particular target-samplecombination T_(j)-P_(k). Further, the anti-target region αT_(j) of therespective solution probe S_(i,j) must be complementary to theparticular target T_(j) of the target-sample combination. For example,if it has been predetermined that the target-sample combination T_(10,k)is to be assembled at capture probe C₅ on the array apparatus 11 for agiven experiment or assay, then the solution probe S which will addressthe target T_(10,k) to capture probe C₅ is the solution probe S_(5,10).The solution probe S_(5,10) has an anti-target region αT₁₀, which iscustomized to bind to the target T₁₀, and has an anti-capture regionαC₅, which is customized to bind to the capture probe C₅. In thisexample, the target T₁₀ can be from all samples P_(1→x) or from onesample P_(k), or target T₁₀ can be one of multiple different targetsT_(1→m) from multiple samples P_(1→x), depending on the type ofmultiplexing assay to be performed.

The plurality of solution probes S of the present assay system 10 arecustomized for each assay. FIG. 2 illustrates the assembly oftarget-sample combination T₄-P₆ or T_(4,6) to the array apparatus 11.The anti-capture region αC₁ of the solution probe S_(1,4) is madecomplementary to the capture probe C₁ at a known location on the array11. Further, the anti-target region αT₄ of that solution probe S_(1,4)is made to be complementary to the target T_(4,6). During the assay, theparticular target T_(4,6), if present in a biological sample P₆ undertest, will hybridize to the particular solution probe S_(1,4) at itsanti-target region αT₄. Moreover, the hybridized target T_(4,6)-solutionprobe S_(1,4) will hybridize via its anti-capture region αC₁ to thecorresponding capture probe C₁ on the array, such that the presence ofthe particular target T₄ in the particular sample P₆ will beascertainable by the presence of the target T_(4,6) on the array 11 atthe known location of capture probe C₁ after the assay. Likewise,multiple different targets T_(1→m) in the same or multiple differentsamples P_(k=1→x), can be assayed along with target T₄ on the same arrayapparatus 11 at capture probes C_(2→n) using respective solution probesS_(2→n. 1→m). The assay system 10 can multiplex one or more biologicalsamples, having one or more biological targets per sample all on thesame array apparatus 11 using the customized solution probes S and thegeneric capture probes C on the array apparatus 11 of the presentinvention. It should be noted that the addressing numbering system usedin the description of the invention herein is illustrative only. Otheraddressing systems could be used, as should be obvious to one skilled inthe art. Therefore, it is within the scope of the present invention touse any appropriate addressing system on the multiplexing array.

The assay method 30 of multiplexing on a single array one or morebiological samples, having one or more biological targets per sample,according to the present invention, is illustrated in FIG. 3. The assaymethod 30 uses the assay system 10, as described above. The assay methodof multiplexing 30 comprises the steps of providing (32) the arrayapparatus 11 comprising the substrate 12 with a first plurality ofbiological probes (capture probes C) in an array pattern on the surface13 of the substrate 12. Each capture probe C_(i) within the firstplurality C is different and each different probe C_(i) is located in adifferent feature location on the array apparatus 11. In the preferredembodiment, there are multiple copies of each different capture probeC_(i) at each feature location on the array 11 to improve the accuracyof the assay.

The assay method 30 of multiplexing further comprises the step ofproviding (34) the second plurality of biological probes, calledsolution probes S for the assay system 10. Each solution probe S_(i,j)of the second plurality S comprises a first region (anti-capture regionαC_(i)) and a second region (anti-target region αT_(j)). As describedabove, the solution probes S are customized to deliver the differenttarget-sample combination to be assayed to their corresponding captureprobes C_(i) on the array (i.e. “addresses”). The first regions αC_(i)of each solution probe S_(i,j) are customized to be complementary torespective different capture probe C_(i) on the array 11. The secondregions αT_(j) of each solution probe S_(i,j) are customized to becomplementary to respective targets T_(j). The customization of thesolution probes S predetermines which capture probe C_(i=1→n) feature onthe array will receive a particular target-sample combination T_(j,k).The customized solution probes S also deliver or assemble the respectivetarget-sample combinations onto the array at their correspondingaddressed capture probe C locations.

The assay method 30 of multiplexing biological materials on a singlearray further comprises the step of assembling (36) one or morerespective target T_(j,k) from the one or more biological samples P_(k)to the array apparatus 11 for evaluation. If present in the particularsample P_(k) under test, the target T_(j,k) is assembled onto the arrayapparatus 11 at capture probe feature C_(i) by the solution probeS_(i,j) via a hybridization process. The hybridization process may besimultaneous or preferably in two hybridization steps. The sequence ofthe hybridization steps or the use of a simultaneous hybridization isnot crucial, except where there are multiple samples being assayed, asfurther described below. However, in the preferred two stephybridization process, the hybridization of the targets T with thesolution probes S in the solution phase is preferred. In onehybridization step (step 36 a), a target T_(j), from a sample P_(k) ispre-incubated with the solution probe S_(i,j) to allow the binding orhybridization to occur in solution between the target T_(j,k) and theanti-target region αT_(j) of the solution probes S_(i,j) usingwell-known conventional methods of hybridization. Preferably, thishybridization step 36 a is performed first. The concentration of thesolution probes S is equivalent to or in excess of the expectedconcentration of the target T. Preferably, the concentration of solutionprobes S is in sufficient excess, such that the free concentration ofthe solution probes S is not decreased more than 10% from binding to itstarget T. After the pre-incubation step (36 a), the other hybridizationstep (step 36 b) includes applying the hybridized {targetT_(j,k)-solution probe S_(i,j)} species in solution to the arrayapparatus 11 under well-known conditions for hybridization or bindingbetween the respective capture probe C_(i) and the anti-capture αC_(i)region of the solution probe S_(i,j) to occur. Alternatively, under somecircumstances, the hybridization steps can be reversed, but as with thesimultaneous hybridization, the advantages of hybridizing the solutionprobes with the target in solution are not available. In this alternatehybridization procedure, the solution probe S_(i,j) can be pre-incubatedwith the capture probe C_(i) on the apparatus 11 (step 36 b′). Then thetarget T_(j,k) is added to the {capture probe-solution probe} system 10for binding to the anti-target region αT_(j) of the solution probesS_(i,j) (step 36 a′). In all situations, the hybridization or binding isperformed under well-known conditions in the art.

The assay method 30 of multiplexing further comprises the step ofwashing (38) the hybridized array apparatus 11 to removeunhybridized/unbound material using conventional materials andprocesses; and the step of analyzing (39) the results of the assay,according to conventional methods.

According to the invention, either the capture probes C, solution probesS and/or the targets T are labeled at any step(s) before or during theassay process to either directly or indirectly produce a signal, such asfluorescence or radiation, for example, using conventional labelingtechniques and materials. The present invention does not require aparticular labeling method or materials and it is believed that any ofthe conventional techniques and materials will work with the invention.The hybridized array 11 is analyzed (39) using conventional equipment,such as optical scanning, to track, sort, identify and/or furthercharacterize the targets T_(1→m) in the samples P_(1→x) assayed.

FIGS. 4A and 4B illustrate the concept of multiplexing in accordancewith the invention. In FIG. 4A, the assay will determine whether aparticular target T₁ is present in multiple samples P_(1→x) (e.g., frommultiple patients). In this embodiment, samples P_(1→x) are kept inseparate vessels. Solution probes S_(1→n,1) are provided, each with adifferent anti-capture αC_(1→n) region corresponding to the differentcapture probe addresses C_(1→n) on the array where it has beenpredetermined that the different samples P_(1→x) will be assembled for agiven assay. Moreover, the solution probes S_(1→n,1) have the sameanti-target region αT₁, because only one target T_(j=1) is being soughtin multiple samples P_(1→x). Only the anti-capture regions αC_(1→n) onthe solution probes S_(1→n,1) distinguish the samples from one another.Therefore, each of the different solution probes S_(1→n,1) are added tothe separate vessels containing the respective samples P_(1→x). Duringthe hybridization step 36 a, sample P₁ is pre-incubated with solutionprobes S_(1,1), and separately, sample P₂ is pre-incubated with solutionprobes S_(2,1), and so on, to the last sample P_(x) being separatelypre-incubated with solution probes S_(n,1). Note that features may beincremented with replicates, control probes, etc., such that n does notequal x. The indexing used herein is just an example and depends uponthe array design, that is, use of replicate features for samples,control samples, etc.

After the hybridization step 36 a is completed, the hybridized speciesmay be mixed together for the second hybridization step 36 b. Duringstep 36 b, the {hybridized targets T_(1,1→x)-solution probes S_(1→n,1)}complexes are incubated with the array apparatus 11. Since the targetsT_(1,1→x) were addressed to be assembled on respective capture probesC_(1→n) by the solution probes S_(1→n,1), solution probes S_(1,1) willassemble the target T_(1,1) on capture probe C₁. Therefore, any targetsT₁ present at probe location C₁, as a result of the hybridization steps36 a, 36 b, originated from sample P₁. The same is true for the targetsT₁ from the other samples P_(2→x) and the other respective captureprobes C_(2→n) and solution probes S_(2→n,1). The system 10 and method30 essentially self-assemble the targets T_(1,1→x) during the assay.After the hybridized array apparatus 11 is washed 38, the assay resultsare analyzed 39. The analysis step 39 will reveal whether and how much,among other things, of target T₁ is present in samples P_(1→x). Thealternative simultaneous hybridization and reversed hybridization steps36 a′ and 36 b′ are not recommended when a target from more than onesample is being assayed.

FIG. 4B illustrates an example of multiplexing multiple differenttargets T_(1→m) from one sample P_(k=1), on a single array according tothe invention. FIG. 4B illustrates a single vessel containing a solutionof multiple (different) targets T_(1→m,1) and different solution probesS_(1→n,1→m). In this example, the solution probes S_(1→n,1→m) have bothdifferent anti-capture regions αC_(1→n), and different anti-targetregions αT_(1→m). The anti-target regions are customized to the targetsT_(1→m,1) under test. The anti-capture regions are customized tocorrespond to the different generic capture probes C_(1→n), in order todeliver the hybridized targets to the appropriate addresses on thearray. The different anti-target regions αT_(1→m) of the differentsolution probes S_(1→n,1→m) will hybridize with their respective targetsT_(1→m,1) in step 36a. The different anti-capture regions αC_(1→n) ofthe different {hybridized target T_(1→m,1)-solution probes S_(1→n,1→m)}species will hybridize with their respective capture probes C_(1→n) onthe array apparatus 11 in step 36 b. Since there is only one sample P₁in this example, whether and how much, among other things, of theparticular targets T_(1→m) are present in the sample P₁ can beascertained by their presence at their respective addressed captureprobe locations C_(1→n) on the array during analysis. Moreover, thealternative simultaneous hybridization or the reversed hybridizationsteps 36a′, 36 b′ can be used in such assays of one sample, as providedin the example illustrated in FIG. 4B.

FIG. 4C illustrates the assay of multiple different targets T_(1→m) frommultiple different samples P_(1→x) using the plurality of solutionprobes S_(1→n,1→m) that each have both different anti-capture regionsαC_(1→n) and different anti-target regions αT_(1→m). The anti-captureregions are customized to correspond to the different generic captureprobes C_(1→n), in order to deliver the hybridized targets to theappropriate addresses on the array. The anti-target regions arecustomized to the targets T_(1→m,1→x) under test. This customization ofsolution probes provides an assembly of the different target-samplecombinations onto the array at pre-determined capture probe locations 14_(1→n) (i.e. “addresses”) for evaluation. The vessels containingrespective samples P_(1→x) must be kept separated, and the differentsolution probes S_(1→n,1→m) customized for the respective samplesP_(1→x) are pre-incubated only with the respective separate samplesP_(1→x). After pre-incubation (step 36a), the hybridized probesS_(1→n,1→m) can be combined for the hybridization step 36 b with thecapture probes C_(1→n) on the array apparatus 11. The alternativesimultaneous hybridization or the reversed hybridization steps 36 a′, 36b′ are not recommended for the example illustrated in FIG. 4C. Thedifferent solution probes S_(1→n,1→m) will sort and assemble thedifferent targets T_(1→m,1→x) from the different samples P_(1→x) ontothe array 11 according to the predetermined addresses provided by thecapture probe locations 14 _(1→n).

As illustrated in FIG. 4C, target T_(1,1) from sample P₁ was addressedand assembled on capture probe C₁ with solution probe S_(1,1), such thatthe presence and quantity of a target T_(1,1) in sample P₁ can beascertained by its presence at its corresponding capture probe locationC₁ on the array during analysis. Moreover, targets T_(6,1) and T_(4,1)from sample P₁ were addressed and assembled on capture probes C₂ and C₃,with solution probes S_(2,6) and S_(3,4), respectively, for evaluation.From sample P₂, targets T_(1,2), T_(3,2) and T_(5,2) were addressed andassembled on capture probes C₄, C₅ and C₆ by solution probes S_(4,1),S_(5,3) and S_(6,5), respectively, for evaluation.

It should be understood that the number of samples and the number oftargets per sample that are multiplexed on a single array 11 with theassay system 10 and method 30 of the present invention are only limitedby the number of different capture probes C present on the arrayapparatus 11. Therefore, the discussion above and examples illustratedin FIGS. 3, 4A, 4B and 4C are merely illustrative of all of the manynumerous different combinations of samples-and-targets that can bemultiplexed on the single array 11, according to the present invention.

The solution probes S of the present invention are unique assay tools inthat they were developed to improve the accuracy of conventionalsandwich hybridization assays so that sandwich hybridization assays canbe performed effectively on arrays. In addition, the solution probes Sof the present invention advantageously expand the usefulness of thesandwich hybridization assay on an array to multiplexing one or morebiological samples having one or more targets per sample on a singlearray. The ability to customize the solution probes S to both biologicaltargets T and to the capture probes C on the array facilitate theassembly of the targets T in predetermined locations on the arraysurface 13 during a given assay and make multiplexing on the arraypossible. Therefore, the set of solution probes S of the presentinvention is a robust tool for multiplexing one or more samples, havingone or more targets per sample on a single addressable andself-assembling array. Moreover, the customization of the solutionprobes S allows the array apparatus 11 and capture probes C to begeneric to the assay and produced in bulk, which is more cost effective.

The processes and materials used for the manufacture of the array system10 and the ancillary materials used for the assay method 30 will dependon the biological materials that are used and analyzed. The system 10 ismanufactured using conventional well-known processes and materials.Table 1 provides a list of biological materials that are useful for thecapture probes C, solution probes S and targets T of the invention. Itshould be understood by those skilled in the art that the presentinvention has broad application to biological material analysis and thatthe present system 10 and method 30 are configurable to accommodate anybiological material, such that the combinations listed in Table 1 areillustrative only. For information regarding the processes and materialsused for the manufacture of arrays of biological materials, such asproteins, antibodies, or the like in accordance with the invention, seefor example U.S. Pat. Nos. 4,591,570; 5,143,854; and 5,252,743 and thefollowing articles: Ekins, R., et. al., “Development of microspotmulti-analyte ratiometric immunoassay using dual fluorescent-labeledantibodies” Analytica Chimica Acta, (1989), 227:73-96; and Ekins, R Pand F W Chu. “Multianalyte microspot immunoassay—microanalytical‘compact disc’ of the future” (1991). There are several common methodsfor attaching oligonucleotides to proteins and ligands, most preferablythe use of biotin-avidin to cross-link the species. Other methods tocross-link species include the use of homobifunctional orheterobifunctional groups (e.g. to cross-link an amine-derivatizedoligonucleotide to a protein). Materials and methods for usingbiotin-avidin, as well as for bifunctional group cross-linking, areavailable from Pierce (Rockford, Ill.). For oligonucleotide synthesis,see the references cited in the Definitions section above.

TABLE 1 Species of Biological Material for Capture Probes, SolutionProbes and Target Samples Capture Anti-Capture Anti-Target Probes RegionRegion Target Sample  1 Oligo- Oligonucleotide cDNA Oligonucleotidenucleotide (e.g., mRNA)  2 Oligo- Oligonucleotide PCR ProductOligonucleotide nucleotide (e.g., mRNA)  3 Oligo- OligonucleotideOligonucleotide Oligonucleotide nucleotide  4 Oligo- OligonucleotideAntibody Antigen nucleotide  5 Oligo- Oligonucleotide Antigen Antibodynucleotide  6 Antigen Antibody Oligonucleotide Oligonucleotide  7Antigen Antibody cDNA or PCR Oligonucleotide Product  8 Antibody AntigenOligonucleotide Oligonucleotide  9 Antibody Antigen cDNA or PCROligonucleotide Product 10 Oligo- Oligonucleotide Receptor Ligandnucleotide 11 Oligo- Oligonucleotide Ligand Receptor nucleotide 12Receptor Ligand Oligonucleotide Oligonucleotide 13 Ligand Receptor cDNAor PCR Oligonucleotide Product

The present system 10 can be packaged as a kit with instructions for usein accordance with the method 30 and provided to users, such as researchand/or analytical laboratories, for practicing the multiplexing assay inaccordance with the invention. The user need only specify the type ofcapture probes they want their kit to contain and the necessaryinformation about the biological materials that the user will beassaying. The solution probes S can be customized to the user'sspecifications. Thus, each solution probe S of a particular set maycomprise first regions selected from oligonucleotides, antibodies,antigens, ligands and receptors, for example, depending on thebiological make-up of the biological features on the array apparatus 11in the kit. Further, the second regions of the set of solution probes Sof the kit may comprise second regions selected from cDNA, PCR products,oligonucleotides, antibodies, antigens, ligands and receptors, forexample, depending on the biological make-up of the biological targetsto be assayed by the user. For example, a user may request a kitcontaining a set of solution probe S, wherein each solution probe S maycomprise a different antigen linked to the same or a different cDNA,wherein the different antigens are complementary to a plurality ofdifferent antibody features on the array apparatus 11 and the cDNA iscomplementary to oligonucleotide (e.g., mRNA) target material(s) to beassayed (see for example, Table 1, examples 8 and 9). The set ofsolution probes S in the kit delivers each target material T beingassayed by the user to a predetermined “address” or capture probe C onthe universal array 11. During the assay, the set of solution probes Swill essentially deliver or assemble each target material T to itscorresponding address to provide an addressable and self-assemblingarray to the user. The kit is particularly useful for multiplexingassays of one or more biological sample(s), having one or morebiological target(s) per sample, on a single array.

In another embodiment of the present invention, an assay system 20 forassaying biological materials that has good specificity and sensitivityis provided. The assay system 20 has good specificity by providing areduced likelihood of cross hybridizations between noncomplementarymaterials. The assay system 20 has good sensitivity by providing areduced likelihood of intramolecular structures within the biologicalprobes. The assay system 20 comprises an assay apparatus 21 that has afirst set of biological material probes, called capture probes 2C, onthe surface 23 of a substrate 22. The capture probes 2C each comprises asequence of monomers. The sequence of monomers in each capture probecomprises one or more modified monomer(s) M, as defined herein andabove, to preferentially hybridizes with complementary similarlymodified monomer(s) M. The set of capture probes 2C is generic to allassays of biological target material.

As mentioned above for the assay system 10, the capture probes 2C forthe system 20 can be synthesized in situ directly onto the substrate 22,added as a intact species which is then covalently linked to thesubstrate 22, or provided using other processes and materials, all ofwhich are within the scope of the invention. The modified monomer M issynthesized into the sequence of monomers using the same conventionalmethods. As a result, the apparatus 21 is universal and generic withrespect to the target material to be tested. The apparatus 21 can bemanufactured in bulk quantities to reduce cost and turn around time.Likewise, the set of capture probes 2C are generic and can bemanufactured in bulk quantities and added to the substrate 22 as neededto reduce cost and turn around time. Customization of the assay system20 is provided by the solution probes 2S, described below.

The assay system 20 having good specificity and sensitivity furthercomprises a second set of biological material probes, called solutionprobes 2S. The set of solution probes 2S are similar to the solutionprobes S of system 10 in that the set of solution probes 2S indirectlylinks or assembles target materials 2T to be tested to the assaysubstrate 22 for analysis. Moreover, there is a different set ofsolution probes 2S for each type of biological material being tested.Customization of an assay is accomplished with the customization of thesolution probes 2S to the biological targets 2T to be assayed and to thecapture probes 2C on the apparatus 21. The solution probes 2S comprise afirst sequence region of monomers, called an anti-capture sequence α2C,that is customized to be complementary to the sequence of monomers inthe capture probe 2C, for hybridizing or binding to the capture probe2C. The set of solution probes 2S further comprises a second sequenceregion, called an anti-target sequence α2T, that is customized to becomplementary to a target 2T under test, for hybridizing or binding tothe target 2T. The two-part customization of the solution probes 2Srenders the system 20 particularly useful for sandwich hybridizationassays.

The set of solution probes 2S is different from the solution probes S ofsystem 10 in that the anti-capture sequence regions α2C comprise one ormore modified monomer(s) M that preferentially hybridize withcomplementary similarly modified monomer(s) M of the capture probes 2C.Therefore, the system 20 has good specificity because the preferentialbinding between the similarly modified monomers M of the complementarycapture probes 2C and the anti-capture sequence regions α2C provides areduced likelihood of cross hybridizations (e.g., hybridizations withmismatches) between noncomplementary sequences. Moreover, the system 20has good sensitivity due to the presence of modified monomers Minterspersed among the unmodified (or not similarly modified) monomers.Since the binding of complementary modified monomers M to unmodifiedmonomers is not favored thermodynamically, there is a reduced likelihoodof intramolecular structures, such as hairpins, forming within thecapture probes 2C or within the anti-capture sequence regions α2C.

As mentioned above for assay system 10, Table 1 lists the biologicalmaterials useful for the system 20 of the present invention withoutlimitation. However, the discussion below will focus on species 1-5 and10-11 of the preferred embodiment, having oligonucleotides for thecapture probe 2C and anti-capture region α2C. Table 1 listsrepresentative anti-target regions, α2T and target 2T sample speciesselected from, but not limited to, oligonucleotides, mRNAs, cDNAs, PCRproducts, antibodies, antigens, ligands and receptors, for species 1-5and 10-11. These and other complementary biological materials will workin the preferred embodiment of the present invention.

In the preferred embodiment, the oligonucleotide capture probe 2C andcomplementary oligonucleotide region α2C of the solution probe 2S aresynthesized with one or more complementary reversed polaritynucleotides, such that the nucleotides with reversed polarity willpreferentially hybridize to complementary reversed polarity nucleotides.The reversed polarity nucleotides preferentially hybridize to each otherrather than to a complementary non-reversed polarity nucleotide becauseit is thermodynamically more favorable, as is further described below.

The term “reversed polarity” used here is the same as the term “invertedpolarity” defined in U.S. Pat. Nos. 5,399,676; 5,527,899; and 5,721,218,mentioned above, which are incorporated herein by reference. Theoligonucleotide probe 2C and region α2C of the invention each containsat least one monomer that has opposite or reversed polarity relative tothe polarity of the growing oligonucleotide sequence. The polarity ofthe growing oligonucleotide sequence is determined, for the purposes ofthe invention, from the polarity of the first synthesized nucleotide ofthe oligonucleotide sequence.

Conventional synthesis of an oligonucleotide is in the (5′→3′)direction, wherein the 5′-end of a growing oligonucleotide chainattaches to a new nucleotide at the new nucleotide's 3′-end duringsynthesis (hereinafter the oligonucleotide is referred to as having a“(5′→3′) polarity”). This conventional synthesis forms (5′→3′)internucleotide linkages between adjacent nucleotides in the sequence.Moreover, it is not uncommon for oligonucleotide synthesis to beperformed in the (3′→5′) direction, wherein the 3′-end of the growingoligonucleotide chain attaches to a new nucleotide at the newnucleotide's 5′-end during synthesis (hereinafter the oligonucleotide isreferred to as having a “(3′→5′) polarity”). The (3′→5′) synthesisdirection forms (3′→5′) internucleotide linkages between adjacentnucleotides in the sequence.

However, what is uncommon, is to synthesize an oligonucleotide with amixture of both synthesis directions, thereby forming oligonucleotidescomprising both (5′→3′) and (3′→5′) synthesis orientations or“polarities” in the same oligonucleotide. As a consequence, theoligonucleotide with the mixture of synthesis orientations comprisesboth heterogeneous (5′→3′) and (3′→5′) internucleotide junctions orlinkages and homogenous (3′→3′) and (5′→5′) internucleotide linkages.Hereinafter the (3′→3′) and (5′→5′) internucleotide linkages are eachreferred to as “an inverted polarity linkage”, since they are formed atthe junction between two nucleotides where there has been a switch fromone polarity to the opposite polarity during synthesis. Moreover, thecombination or mixture of polarities in a nucleotide sequence is notnormally found in nature. Therefore, not wanting to be limited to oneconventional synthesis direction or the other, one way to define a“reverse polarity nucleotide {overscore (N)}”, for the purposes of thisinvention, is a nucleotide that has an “opposite” or a “reverse”polarity (i.e., reversed synthesis direction) relative to theorientation of the first, or of other nucleotide(s) N in theoligonucleotide sequence. Moreover, an oligonucleotide sequencecomprising a mixture of polarities further comprises at least one(3′→3′) or (5′→5′) homogenous inverted polarity linkage.

FIG. 5 illustrates an example of the preferred embodiment of the presentinvention utilizing reverse polarity nucleotides {overscore (N)} in anoligonucleotide capture probe 2C and oligonucleotide anti-capture α2Cregion of a solution probe 2S of system 20. The capture probe 2C isattached to the surface 23 of the substrate 22. For simplicity, onlyfive nucleotides of the capture probe 2C and anti-capture region α2C areshown in some detail for the purposes of the following discussion. Thefirst nucleotide of the oligonucleotide sequence illustrated in FIG. 5is nucleotide A₁. This first nucleotide A₁ has the conventional 5′→3′orientation, that is, the 5′ end of the nucleotide A₁ is “up” and isready for synthesis in the conventional 5′→3′ direction. Whether anyother nucleotide in the sequence has a reversed polarity will depend onwhether the orientation of the other nucleotide(s) is the same orreversed relative to the orientation of nucleotide A₁. The secondnucleotide T₁ has a 3′→5′ synthesis orientation. Therefore, nucleotide{overscore (T)}₁ has a reversed polarity relative to the polarity of theoligonucleotide sequence. Moreover, nucleotide G₁ has a 3′→5′orientation. Therefore, nucleotide {overscore (G)}₁ also has a reversedpolarity orientation relative to the orientation of nucleotide A₁. Thenext two nucleotides, C₁ and A₂, have 5′→3′ orientations, and therefore,have the same orientation as the first nucleotide A₁ of theoligonucleotide sequence.

The reversed polarity nucleotides {overscore (N)} may be groupedtogether and/or interspersed throughout the oligonucleotide sequencebetween the same polarity nucleotides N, as is further discussed below.A reverse polarity nucleotide segment ({overscore (N)} s) comprises agroup or sequence of s adjacent reverse polarity nucleotides {overscore(N)}, wherein s is one or more nucleotides {overscore (N)}. The segment({overscore (N)} s) further comprises either a (3′→3′) or a (5′→5′)inverted polarity linkage at one end of the segment ({overscore (N)} s)that links with a same polarity nucleotide N. If the segment ({overscore(N)} s) is linked at the other end by a same polarity nucleotide N, thenthe segment ({overscore (N)} s) will further comprise the other of theinverted polarity linkages at that end of the segment. FIG. 5illustrates the segment ({overscore (N)} s) of {overscore(T)}₁-{overscore (G)}₁ in the oligonucleotide probe 2C bounded by samepolarity nucleotides A₁ and C₁. Therefore, the segment ({overscore (N)}s) has both (3′→3′) and (5′→5′) inverted polarity linkages at either endof the segment ({overscore (N)} s).

Also illustrated in FIG. 5 is the complementary solution probe 2S of thesystem 20. The complementary anti-capture region α2C of the solutionprobe 2S is hybridized to the capture probe 2C in the conventionalanti-parallel relationship. Since the capture probe 2C has been definedabove as being in a 5′→3′ synthesis orientation, the anti-parallelcomplementary anti-capture region α2C is by definition in the 5′→3′synthesis orientation, starting at the anti-target region α2T through T₁of the anti-capture region α2C. The nucleotides T₁, G₁, and T₂ of theanti-capture oligonucleotide sequence region α2C have a 5′→3′ synthesisorientation and therefore, are same polarity nucleotides N. Thenucleotides T₁, G₁, and T₂ are hybridized to same polarity nucleotides,A₁, C₁, and A₂ in the capture sequence 2C, respectively. The nucleotides{overscore (A)}₁ and {overscore (C)}₁ in the anti-capture sequence α2Cboth have a 3′→5′ synthesis orientation and therefore, are reversedpolarity nucleotides. The reverse polarity nucleotides {overscore (A)}₁and {overscore (C)}₁ are hybridized to the reversed polarity nucleotides{overscore (T)}₁ and {overscore (G)}₁ in the capture sequence 2C,respectively.

The reversed polarity nucleotides {overscore (A)}₁ and {overscore (C)}₁in the anti-capture sequence region α2C also form a reversed polaritynucleotide segment ({overscore (N)} s) that is hybridized to the segment({overscore (N)} s) in the capture probe 2C. The reversed polaritynucleotide segment ({overscore (N)} s) in the anti-capture sequence isbounded on either side by same polarity nucleotides N; thus, the segment({overscore (N)} s) has both (5′→5′) and (3′→3′) inverted polarityinternucleotide linkages at either end as illustrated in FIG. 5.

Also illustrated in FIG. 5 is an anti-target region α2T of the solutionprobe 2S in accordance with the invention. The anti-target region α2T isattached or linked to the anti-capture region α2C and independently to arespective target 2T as the result of an assay.

The number of total nucleotides, the number of reverse polaritynucleotides {overscore (N)} or segments {overscore (N)} s in the probes2C, 2S shown in FIG. 5 are illustrative only and in no way is intendedto limit the scope of the invention. The total number of nucleotides andnumber of reverse polarity nucleotides {overscore (N)} or segments{overscore (N)} s in the capture probes 2C and anti-capture regions α2Cthat are within the scope of the invention are described below.

The reverse polarity nucleotides {overscore (N)} used in accordance withthe invention are described in more detail in:

(1) M. Koga, M. Moore and S. Beaucage, “Alternatingα,β-Oligothymidylates with Alternating (3′→3′)-and(5′→5′)-Internucleotidic Phosphodiester Linkages as Models for AntisenseOligodeoxyribonucleotides”, The Journal of Organic Chemistry, 1991,Volume 56, No. 12, pp. 3757-3759;

(2) M. Koga, S. Geyer, J. Regan and S. Beaucage, “The synthesis ofalternating α,β-oligodeoxyribonucleotides with Alternating (3′→3′)- and(5′→5′)-Internucleotidic Linkages as Potential Therapeutic Agents”,Nucleic Acids Symposium Series, No. 29, 1993, pp. 3-4; and

(3) M. Koga, A. Wilk, M. Moore, C. Scremin, L. Zhou, and S. Beaucage,“Synthesis and Physiochemical Properties of Alternatingα,β-oligodeoxyribonucleotides with Alternating (3′→3′)- and(5′→5′)-Internucleotidic Phosphodiester Linkages”, J Org. Chem., 1995,60, 1520-1530,

each of which is incorporated herein by reference.

A nucleotide with reversed polarity {overscore (N)} in anoligonucleotide sequence hybridizes preferentially with another reversepolarity complementary nucleotide {overscore (N)}, because the bond isthermodynamically more stable than a bond between a reversed polaritynucleotide {overscore (N)} and a complementary non-reversed polaritynucleotide N. The hybridization bond stability is measured as a functionof denaturation temperature “Tm”. The inverted polarity (3′→3′) and(5′→5′) linkages at either end of the reverse polarity nucleotide{overscore (N)} or segments {overscore (N)} s are believed to havedifferent internucleotide bond-lengths when compared with theconventional (3′→5′) and (5′→3′) linkages and this disparity ininternucleotide distances impairs the hybridization between reversepolarity {overscore (N)} and non-reversed polarity nucleotides N (seeReferences (2) and (3) above).

For the purpose of emphasizing the thermodynamic effect that the reversepolarity nucleotides {overscore (N)} have in providing good specificityand sensitivity of the system 20 in accordance with the invention, someof the evaluation and results reported by M. Koga et al. in References(2) and (3) are summarized below. The denaturation temperatures (Tm's)were evaluated and reported by M. Koga et al. for the hybridization ofreversed polarity oligomers having 24 nucleotides in length(α,β-oligonucleotides) that were complementary to an unmodified regionoverlapping the splice acceptor site of the second exon encoding theHIV-1 Tat gene product. The α,β-deoxy-oligonucleotides had alternating(3′→3′) and (5′→5′) phosphodiester internucleotide linkages (“modifiedd-oligomers₂₄”). The complementary unmodified deoxy-oligonucleotidescomprised only natural (5′→3′) phosphodiester linkages (“naturald-oligomers₂₄”). The different combinations of natural and modifieddeoxy-oligomer duplexes are reproduced as No. 1-3 in Table 2 below. Alsoevaluated and reported by M. Koga et al. were the Tm's for theribo-oligonucleotides (“natural r-oligomer₂₄”) hybridized with themodified and natural d-oligomers₂₄. The Tm's of the deoxy- andribo-oligomer duplexes also are reproduced in No. 4-5 in Table 2 below.

TABLE 2 Tm of Natural and Modified Polynucleotide Duplexes (Informationherein is reproduced in part from Table 1 of Ref. 2 (for No. 1, 2, 3below) and Table 3 of Ref. 3 (for No. 4, 5 below)) No. Duplex Tm Δ 1natural d-oligomer₂₄ - natural d-oligomer₂₄ 67°C. — 2 modifiedd-oligomer₂₄ - natural d-oligomer₂₄ 53°C. −14°C. 3 modifiedd-oligomer₂₄- modified d-oligomer₂₄ 61°C.  −6°C. 4 naturald-oligomer₂₄-natural r-oligomer₂₄ 62°C. — 5 modifiedd-oligomer₂₄-natural r-oligomer₂₄ 35°C. −27°C.

The denaturation temperatures are 14 degrees lower for duplexes of“natural-modified” oligonucleotide duplexes (No. 2) than for the“natural-natural” oligonucleotide duplexes (No. 1), which indicates alack of stable hybridization or binding between complementary naturaland reverse polarity nucleotides. However, the Tm for the duplex of“modified-modified” oligonucleotides (No. 3) was 8 degrees higher thanthe “natural-modified” oligonucleotides (No. 2) and only 6 degrees lowerthan the “natural-natural” oligonucleotide (No. 1), which indicates thatthe hybridization bonds are stronger and thermodynamically more stablefor the “modified-modified” nucleotides than their “modified-natural”oligonucleotide counterparts. Therefore, the reversed polaritynucleotides {overscore (N)} of the system 20 will be less likely tocross-hybridize with other complementary nucleotides N havingnon-reversed polarity. Advantageously, the reversed polarity nucleotides{overscore (N)} of the system 20 will be even less likely tocross-hybridize with noncomplementary or “mismatched” nucleotides N. Thechemically modified nucleotides {overscore (N)} effectivelypreferentially hybridize with complementary similarly modifiednucleotides {overscore (N)}.

The effect is believed to be more disruptive to cross-hybridizationsthan would be observed from a natural or non-reversed polaritynucleotide N mismatch, as provided in the conventional methods ofminimizing cross-hybridizations (see Brenner references). Thus,cross-hybridization between a sequence containing a reversed polaritynucleotide {overscore (N)} or segment {overscore (N)} s, in accordancewith the invention, and a non-reversed polarity nucleotide N or sequenceNs, such as those also present in the capture probes 2C, anti-captureregions α2C, anti-target regions α2T and target samples 2T of thepresent invention, is less likely to occur. Advantageously,intramolecular structures, such as hairpins, are also less likely tooccur, rendering the system 20 of the present invention a powerful androbust tool for diagnostic biological assays.

The reversed polarity nucleotides {overscore (N)} can be obtained asphosphoramidite derivatives, for example, from Glen Research ofSterling, Va. As mentioned above, the capture probes 2C and proberegions α2C of the solution probes 2S can be synthesized with anysynthesis method and preferably, are synthesized in accordance withconventional oligonucleotide synthesis methods. Advantageously, thephosphoramidite derivatives {overscore (N)} are incorporated into theoligonucleotide sequence using these conventional methods.

For the invention, the total length or number (L) of monomers (includingboth unmodified monomers and modified monomers M) in the capture probes2C and anti-capture regions α2C of the solution probe 2S is sufficientenough to form strong hybridizations or binding between complementarycapture probe 2C and anti-capture regions α2C to withstand theconventional stringent washing steps after hybridization. Preferably,the total number or length L of the capture probes 2C and anti-captureregions α2C of the solution probes 2S may be anywhere from 5 to 50monomers, more preferably 10 to 30 monomers in length L, and even morepreferably, between 10 to 25 monomers in length L.

In each of the capture probes 2C and anti-capture regions α2C of thesolution probes 2S illustrated in FIG. 5 of the preferred embodiment,two reversed polarity nucleotides {overscore (N)}, (or one reversepolarity segment {overscore (N)} s), are shown for illustrative purposesonly. In accordance with the invention, the number of modified monomerspresent in a capture probe 2C and solution probe 2S should be highenough to provide a reduced likelihood of cross-hybridizations andintramolecular structures, but not too high so that the number ofmodified monomers does not impact the synthesis yield of the captureprobes 2C or anti-capture regions α2C. The number of modified monomersin a particular capture probe 2C and anti-capture sequence α2C can bereadily determined empirically, without undue experimentation by oneskilled in the art, and depends on the synthesis method chosen.

Generally, there may be anywhere from one to (L−1) modified monomers M,where L is the length of (or total number of overall monomers in) therespective capture probe 2C or anti-capture region α2C; or preferably,from 1 to (⅔)L modified monomers M; and more preferably, (¼)L to (½)Lmodified monomers M in the capture probes 2C and anti-capture regionsα2C of the solution probes 2S. The modified monomers M can beinterspersed in the probe individually (s in {overscore (N)} s=1), orinterspersed in groups or segments (where s>1), as mentioned above. Whenthe modified monomers M are interspersed in a regular pattern, forexample, every other one, the number of inverted polarity linkages areadvantageously maximized. A high number of inverted polarity linkages isadvantageous, since these linkages disrupt cross-hybridizations andintramolecular structures, as discussed above. However, the number ofavailable unique sequence combinations is more limited. If the modifiedmonomers are interspersed in a random pattern, for example, there aremore available unique sequence combinations. If the modified monomersare grouped together in one or more segments, the number of invertedpolarity linkages is not maximized and there are relatively feweravailable unique sequence combinations. One skilled in the art is ableto determine the optimum distribution of modified monomers in a sequencein accordance with the invention.

According to a preferred embodiment, a capture probe 2C, andanti-capture region α2C can be designed with modified monomers M inaccordance with the invention using the following formula: x=L−6−D,where x equals the maximum number of modified monomers M in the probe orregion; L equals the length or total number of monomers of the probe orregion; and 6 is the total number of monomers from the two ends of theprobe or region before a modified monomer is inserted. For the preferredoligonucleotide embodiment, the number 6 represents the total of threenucleotides adjacent to the 3′ end and three nucleotides adjacent to the5′ end of the oligonucleotide probe or region, because empirical resultsshow that the last three nucleotides on the 3′ end and on the 5′ end arenot as involved in specificity. That is, mismatches on the ends are notas disruptive as in the middle of the probe. Further, D equals thenumber of locations that a modified monomer M should differ in each ofthe probes or regions of a set. It is preferred that the locations ofthe reversed polarity nucleotides {overscore (N)} or nucleotide segments{overscore (N)} s in any 2 oligonucleotide probes of the same set shoulddiffer in at least D=2 positions to minimize hybridizations betweennoncomplementary reversed polarity nucleotides {overscore (N)}. Themaximum number x of modified monomers M in a L=24 monomer length captureprobe 2C and anti-capture region α2C, for example, would have at mostx=24−6−2=16 chemically modified monomers M, located in positions p₄ top₂₁ of the total positions p₁ to p₂₄ of the 24 monomer long polymer.Preferably, there are a minimum of two (2) modified monomers M in anyprobe or anti-capture region of the invention. Moreover, it is preferredthat the chemically modified monomers M be dispersed throughout thelength of the probe 2C, and region α2C, rather than grouped together inone location on each and more preferably, dispersed in a random pattern.One skilled in the art would know how to disperse the chemicallymodified monomers M throughout the probe or region length L to achieveoptimum results.

Also, as mentioned above, there should be a sufficient number ofmonomers in the probe and region sequences such that the hybridizationbetween a capture probe 2C and an anti-capture region α2C is strongenough to withstand the stringent well-known washing steps, so that therespective assembled target 2T remains assembled to the apparatus 21 viathe solution probe 2S. The capture probe 2C and anti-capture region α2Cmay each further comprise a member of a specific binding pair Y-Z. Thespecific binding pair Y-Z is cross-linked with a covalent bond or astrong non-covalent interaction that will withstand the stringentwashing steps and help to hold the hybridized capture probes 2C andanti-capture regions α2C together. The strong bond between thecross-linking pair Y-Z is formed using, for example, a chemical catalystor photo-reactive chemistry with illumination. The specific binding pairY-Z of this embodiment would have to be of a type that did not interferewith the base pairing and base-stacking of its neighboring monomers inthe sequence.

FIG. 6 illustrates the specific binding pair Y-Z in this embodiment ofsystem 20. Capture probe 2C, containing the binding member Y, ishybridized to the anti-capture region α2C, containing the binding memberZ, of solution probe 2S. Solution probe 2S is also hybridized or boundto a target 2T at the anti-target region α2T, as illustrated in part (a)of FIG. 6. The members Y and Z are cross-linked, resulting in Y-Z linkedpair, as illustrated in part (b) of FIG. 6. The cross-linked pair Y-Zprovides further assurance that capture probe 2C retains itshybridization bonds to the solution probe 2S after the stringent washingstep, and therefore, that only the specific binding of the appropriatetarget 2T to the respective solution probe 2S is subsequently measuredor analyzed. As a result, this embodiment provides stronger bondsbetween respective capture probe 2C and anti-capture region α2C, and isespecially useful in embodiments where relatively short capture probes2C and anti-capture regions α2C (i.e., less than 15-monomers in totallength L) are desired, but are not able to provide enough hybridizationbonds, in the absence of such cross-linking, to withstand the stringentwashing steps after the hybridization steps.

The capture probes 2C and anti-capture regions α2C monomer sequences mayfurther comprise other monomers, such as polyethylenes orprotein-nucleic acid PNA hybrids, to serve as spacers in the monomersequences. These spacers are added to the capture probes 2C andanti-capture regions α2C using conventional phosphoramidite ornonphosphoramidite techniques, for example. The use of spacers is wellknown in the art for achieving length without specificity, to minimizecross-hybridizations, to distance a reaction from the surface of asubstrate and/or to decrease cost, for example. The above uses areillustrative and in no way intended to limit the scope of the invention.There are many other uses for spacers not mentioned here that arereadily apparent to one skilled in the art. All of which are within thescope of the invention.

The present system 20 with good specificity and sensitivity isparticularly useful in sandwich hybridization assays on arrays andprovides a powerful feature to the system 10 and method 30 formultiplexing one or more samples, having one or more targets per sample,on a single array.

In the preferred embodiment, an array assay system 20″ having goodspecificity and sensitivity for such multiplexing is provided. Thepreferred array system 20″ differs from system 20 by comprising an arrayapparatus 21″ that has the first set of probes 2C located on a substrate22″ in an array of features 24 _(i=1,n), wherein a different captureprobe 2C_(i), is located on a different feature location 24 _(i) of thearray apparatus 21″. There may be multiple copies of each differentcapture probe 2C_(i), at each location. Each capture probe 2C_(i) in theset may be different by having a different sequence of monomers. As insystem 20, the capture probes 2C comprise one or more chemicallymodified monomer(s) M and may comprise a member of cross-linking pairY-Z and/or a spacer, in accordance with the invention.

Moreover, in accordance with this array assay system 20″ embodiment,each solution probe 2S_(i,j), in the second set of probes 2S may bedifferent by comprising a different anti-capture sequence region α2C_(i)and either the same anti-target region α2T_(j) or a differentanti-target region α2T_(j=1→m), depending on the biological materials tobe assayed (i.e., the number of samples P and the number of targets 2Tper sample). The anti-capture sequence region α2C_(i) comprises one ormore chemically modified monomer(s) M that are complementary torespective similarly modified monomers M in the capture probe 2C_(i) andmay also comprise a member of a cross-linking pair Y-Z. There may bemultiple copies of each different solution probe 2S_(i,j), in the set.The set of capture probes 2C provides an address for each targetmaterial 2T_(j,k) on the array and the set of solution probes 2Sessentially assembles or delivers each target 2T_(j,k) to itscorresponding capture probe location 2C_(i) during the array assay toprovide an addressable and self assembling assay with good specificityand sensitivity.

A method 40 of assaying biological materials having good specificity andsensitivity according to the present invention is illustrated in FIG. 7.The assay method 40 comprises the steps of providing (42) the assayapparatus 21 having the first set of biological probes (capture probes2C) comprised of a sequence of monomers attached to the substrate 22.

In the preferred embodiment, an array assay method 40″ is performedusing the array apparatus 21″. Therefore, in the preferred embodiment,the set of capture probes 2C are attached to the surface 23″ of thearray substrate 22″ in an array of feature locations 24. There is adifferent probe 2C_(i) of the set at each different feature location 24_(i). Preferably, there are multiple copies of each different captureprobe 2C_(i) at each feature location 24 ₁.

The method 40, 40″ further comprises the step of providing (44) thesecond set of biological probes (solution probes 2S), that comprises thefirst sequence region of monomers and a second region. The firstsequence region (anti-capture region α2C) of the solution probes 2S iscomplementary to the monomer sequence on the capture probe 2C, and thesecond sequence region (anti-target region α2T) on the solution probes2S is complementary to biological targets 2T to be assayed. In thepreferred array assay embodiment, each solution probe 2S_(i,j), of theset of solution probes 2S comprises the first sequence region α2C_(i)complement to a respective capture probe 2C_(i) of the array apparatus21″ and a second region α2T_(j) complement to a respective target T_(j)in a respective sample P_(k). Therefore, one or more biological samplesP_(k), having one or more biological targets 2T_(j), per sample, can beassayed together (multiplexed) on the same array apparatus 21″.

Each capture probe 2C_(i) of the set of capture probes 2C comprises inthe monomer sequence one or more monomer(s) M having a chemicalmodification. Each first sequence region α2C of the set of solutionprobes 2S comprises one or more respective complementary monomer(s) Mhaving a similar chemical modification, wherein the complementarysimilarly modified monomers M in the capture probes 2C and theanti-capture sequence region α2C of the solution probe 2S willpreferentially hybridize or bind to each other instead of to acomplementary monomer that is not similarly modified (also referred toas an “unmodified monomer”).

When the capture probes 2C and anti-capture regions α2C of the solutionprobes 2S comprise oligonucleotides, as in species 1-5, 10 and 11 ofTable 1, the monomer sequence of the capture probe 2C and theanti-capture monomer sequence region α2C each comprises complementaryreversed polarity nucleotides {overscore (N)} relative to the polarityof the respective monomer sequence, as defined above by the firstnucleotide in the sequence. As stated above for the preferred embodimentof the system 20, “preferentially hybridize or bind to each other” meansthat the reversed polarity nucleotide {overscore (N)} forms athermodynamically more stable hybridization with another complementaryreversed polarity nucleotide {overscore (N)} than with non-reversedpolarity nucleotide N.

The method 40, 40″ of assaying biological materials further comprisesthe step of assembling (46) the target materials 2T to the substrate 22,22″ for evaluation. The target materials 2T are linked indirectly orassembled to the apparatus 21, 21″ similarly to the method 30 describedabove. The hybridizations may be performed simultaneously or preferably,in steps. In the first hybridization step 46 a, the targets 2T arepre-incubated with the solution probes 2S to allow the binding orhybridization to occur in solution phase between the targets 2T and theanti-target regions α2T of the solution probes 2S. The concentration ofsolution probes 2S is preferably the same as that for method 30. Afterthe pre-incubation step (46a), the hybridized {target-solution probe}species in solution is incubated with the apparatus 21, 21″ of captureprobes 2C on the substrate 22, 22″ for hybridization or binding betweenthe capture probes 2C and the anti-capture regions α2C of the hybridized{target-solution probe} species to occur (step 46 b). Alternatively, thehybridization steps can be reversed, but like the simultaneoushybridizations, the advantages of hybridizing the 5 solution probes withthe target in solution are not available. When the hybridizations stepsare reversed, the solution probes 2S can be pre-incubated with thecapture probes 2C on the apparatus 21, 21″ (step 46 b′ dashed arrow inFIG. 7). Then the targets 2T are added to the {capture probes-solutionprobes} system 20, 20″ for binding to the anti-target region α2T of thesolution probes 2S (step 46 a′ dashed arrow in FIG. 7). In allsituations, the hybridization or binding is performed under well-knownconditions in the art. However, the simultaneous hybridizations and thereversed hybridization steps 46 b′, 46 a′ are not recommended where theassay involves one or more biological target(s) 2T_(j,k) in more thanone biological sample P_(k=1 to x), because of the need to keep thesamples separated until the solution probes 2S are hybridized withrespective targets 2T in the respective samples P.

The method 40, 40″ further comprises the step of removing (48) theunhybridized/unbound material from the incubated apparatus 21, 21″ bywashing the apparatus 21, 21″ using conventional materials andprocesses; and the step of analyzing (49) the results of the assay,according to conventional methods. At some point during the assay, alabeling system is added to either the target samples 2T, capture probes2C, solution probes 2S or the like, that produces a signal wheninterrogated during the analysis step 49. Preferably, the label producesan optical signal that is detected optically with conventional opticalscanning equipment, however other labels and detection methods areapplicable to the invention. The labeling system, its introduction intothe assay, and the method of detection are not the subject of thepresent invention. Any conventional labeling and detection techniques,materials and equipment can be used. The hybridized system 20, 20″ isanalyzed (49) to track, sort, identify and further characterize thetargets 2T using conventional equipment.

However, when the capture probes 2C and solution probes 2S comprise thecross-linking pair Y-Z, before the step of removing (48), the method 40,40″ further comprises the step of cross-linking (47) the monomer pairY-Z on the capture probes 2C and solution probes 2S, respectively, tostrengthen the bond or hybridization between the capture probes 2C andrespective anti-capture region α2C on the solution probes 2S to betterwithstand the stringent washing step (48). The step of cross-linking 47is illustrated in FIG. 7 via dashed box and arrow. In the step ofcross-linking 47, preferably, the apparatus 21, 21″ is first washed witha conventional low stringency wash procedure before the specific bindingpair is cross-linked. The Y-Z specific binding pair will cross-link witha covalent bond or a strong non-covalent bond under appropriateconditions, such as photo-activation and/or chemical catalyticactivation after the low stringency wash.

As mentioned above, the assay system 20, 20″ and method 40, 40″ of thepresent invention are advantageously useful for sandwich hybridizationassays, and in particular in sandwich hybridization assays on arrays21″. The system 20, 20″ and method 40, 40″ provide specific andsensitive powerful assay tools for multiplexing one or more sample(s),having one or more target(s) per sample, and that are addressable andself-assembling, as in system 10 and method 30, described above. Thesolution probes 2S are customized for respective targets 2T inrespective samples P and customized for respective capture probes 2C onthe array apparatus 21″. The solution probes 2S will bind or hybridizeto the respective targets 2T and bind or hybridize to respective captureprobes 2C with good specificity and sensitivity due to the preferentialhybridization between complementary similarly modified monomers M.Therefore, the system 20, 20″ and method 40, 40″ provide good accuracyto the assay, which renders them particularly advantageous formultiplexing a plurality of different samples P, each sample P_(k)having a plurality of different targets 2T_(j) per sample, on the samearray 21″.

The system 20, 20″ and method 40, 40″ with complementary modifiedmonomers M provide many advantages, at least three are described below.First, the complementary modified monomers M provide good specificity tothe assay by systematically providing a reduced likelihood of undesiredhybridizations from occurring that are likely reduced relative to system10 and method 30. The likelihood of undesired hybridization is reducedbecause the modified monomers M preferentially hybridize to each otherinstead of with a complementary unmodified monomer. For the preferredembodiment, the hybridization preference of reversed polaritynucleotides {overscore (N)} with each other is due to the disruptiveeffects of the different internucleotide linkage distances of thereversed polarity nucleotides {overscore (N)} and non-reversed polaritynucleotides N. FIG. 8A illustrates one example of a correcthybridization in accordance with the system 20, 20″ of the presentinvention. FIGS. 8B and 8C illustrate two scenarios having mismatchedcross-hybridizations and FIG. 8D illustrates an intramolecularstructure. These figures illustrate problems that are typical inconventional sandwich hybridization assays and which may occur in thesystem 10 and method 30 for multiplexing of the invention describedabove, when modified monomers M are not included to provide goodspecificity and sensitivity.

FIG. 8A illustrates a portion of apparatus 21, 21″ comprising aparticular capture probe 2C₁ located at feature 24 ₁ on the surface 23,23″ the substrate 22, 22″. The capture probe 2C₁ has three (3) modifiedmonomers M, or in the preferred embodiment, reversed polaritynucleotides {overscore (N)}, illustrated by way of example only. Thecapture probe 2C₁ is hybridized to its complementary anti-capture regionα2C₁ of solution probe 2S_(1,1). The solution probe 2S_(1,1) furthercomprises an anti-target region α2T₁ that is complementary andhybridized to a target 2T_(1,x) from an arbitrary sample P_(x), notshown.

Symbolic discrimination and other screening methods are typicallyemployed in conventional systems to minimize cross-hybridizations. Asmentioned above, Brenner discloses one method of symbolic discriminationby introducing at least two mismatched pairs into the polynucleotidetags. Other methods of screening are described in EP 0 799 897 A1 (D.Schoemaker) published Oct. 8, 1997 and U.S. Pat. No. 5,556,749(Mitsuhashi et al.) issued Sep. 17, 1996, among others, all of which areincorporated herein by reference. Each of these methods and others notlisted here would work to screen the capture probes C and anti-captureregions αC of system 10 to produce “minimally cross-hybridizing sets”.These methods of screening would work on the capture probes 2C andanti-capture regions α2C of system 20 as well. However, the conventionalscreening methods are not entirely successful in preventing the problemof cross-hybridizations and some do not address the problem associatedwith intramolecular structures, which affect the assay specificity andsensitivity, respectively.

Advantageously, the system 20, 20″ and method 40, 40″ systematicallyprovide a reduced likelihood of cross-hybridizations and intramolecularstructures even before the probes 2C and regions α2C are screenedaccording to any of the conventional methods. In fact, the system 20,20″ and method 40, 40″ of the present invention are more likely toprevent mismatches between complex targets and the capture probe 2C oranti-capture region α2C during an assay. The conventional screeningmethods are not able to screen for mismatches with complex targets,because complex targets are difficult to screen, especially when thesequences of the target material is not known or not known completely orwith certainty. Consider a target 2T comprised of RNA, for example, andthe respective capture probes 2C and anti-capture regions α2C of thesolution probes 2S used in the assay, each comprising complementaryreversed polarity deoxyribonucleotides {overscore (N)} in theirsequences. According to Table 2 above, advantageously, a mismatchbetween complementary ribonucleotides of the RNA target and thedeoxyribonucleotides {overscore (N)} of the capture probes 2C andregions α2C are even less likely to occur, due to the more disruptiveeffects of the different internucleotide linkage distances of thereversed polarity deoxyribonucleotides {overscore (N)} with respect tonon-reversed or “natural” ribonucleotides of the target. The moredisruptive effects are shown by the ΔTm for No. 5 of Table 2, which is−27° C. for the modified d-oligomer₂₄-natural r-oligomer₂₄ (almost twiceas high as the ΔTm (−14° C.) for the modified/natural d-oligomers, No.2). This advantage of the present system 20, 20″ and method 40, 40″ isevident even when the capture probes 2C and anti-capture regions α2C arethe oligoribonucleotides and comprise reversed polarity ribonucleotides{overscore (N)} and the target to be assayed is a complex DNA, e.g.,cDNA. Therefore, the present invention systematically provides a reducedlikelihood of cross-hybridizations with the target 2T, which issomething that conventional methods are unable to do.

FIG. 8B illustrates an example of a system 10 solution probe S_(2,2)that is linked to an appropriate target T_(2,x), from an arbitrarysample P_(x), but its anti-capture region αC₂ has hybridized to thewrong capture probe C₁ on the substrate 12. The cross-hybridization(hybridization with mismatches) between the monomers of capture probe C₁and the monomers of anti-capture region αC₂ prevents solution probeS_(1,1), comprising the anti-target region αT₁ and the anti-captureregion αC₁, from properly hybridizing to capture probe C₁. Since thisexample has no target T₁ in sample P_(x), the incorrect presence oftarget T_(2,x) at the C₁ address produces a false positive result in anassay for target T_(1,x).

In FIG. 8C, the solution probe S_(2,2) has cross hybridized with captureprobe C₁, at solution probe S_(2,2)'s anti-target region αT₂, such thatthe solution probe S_(2,2) is not able to bind to its appropriate targetT_(2,x), (arbitrarily from sample P_(x)). As a result, the solutionprobe S_(1,1) with its appropriate target T_(1,1) attached (arbitrarilyfrom sample P₁) is blocked, by solution probe S_(2,2), from correctlyhybridizing with the capture probe C₁. This produces a false negativeresult for target T_(1,1). The many different cross hybridizations(hybridization with mismatches) that occur in conventional systems, andthat are possible in the multiplexing system 10 of the invention, aretoo numerous to describe all of them herein. The examples shown in FIGS.8B and 8C are just illustrative of the types of cross-hybridizationsthat could occur. The present system 20, 20″ and method 40, 40″systematically provide a reduced likelihood of thesecross-hybridizations from occurring as a result of the good specificityintroduced by the use of chemically modified complementary monomers M.

The system 20, 20″ and method 40, 40″ provide a reduced likelihood ofhybridization mismatches between (1) capture probes 2C and sequencesthat are not complementary to the capture probes 2C, such asnoncomplementary anti-capture sequences α2C of solution probes 2S,anti-target sequences α2T of solution probes 2S, or target sequences 2T;and (2) anti-capture sequences α2C and sequences that are notcomplementary to the anti-capture sequences, such as noncomplementarycapture probes 2C, other anti-capture sequences α2C, anti-targetsequences α2T of solution probes 2S, or target sequences 2T. Minimizingthe likelihood of cross-hybridizations enhances the specificity of thesystem 20, 20″ and method 40, 40″.

Second, the complementary modified monomers M of the system 20, 20″ andmethod 40, 40″ of the present invention provide a reduced likelihood ofintramolecular structures forming within capture probes 2C and theircomplementary anti-capture sequences α2C. A reduced probability ofintramolecular structures forming increases the sensitivity of thehybridization between the capture probes 2C and their complementaryanti-capture sequences α2C. FIGS. 8D illustrates a solution probe, suchas S_(1,1) of the system 10, wherein an intramolecular structure I hasformed within the anti-capture region αC₁ that hinders the properhybridization between the complementary capture probe C₁ and theanti-capture region αC₁. The intramolecular structure causes a falsenegative for target T_(1,x) (arbitrarily from sample P_(x)). System 20,20″ and method 40, 40″ provide modified monomers M interspersed amongthe sequences of the capture probes 2C and anti-capture regions α2C. Thebinding of modified monomers M to complementary monomers that are notsimilarly modified is not favored thermodynamically. Thus, there is areduced likelihood of intramolecular structures I forming within thecapture probes 2C or within the anti-capture regions α2C. In thepreferred embodiment, the reversed polarity nucleotides {overscore (N)}systematically provides a reduced likelihood that intramolecularstructures I will occur due to the disruptive effects of the differentinternucleotide linkage distances of the reverse polarity nucleotides{overscore (N)} and the non-reversed polarity nucleotides N.

Third, the system 20, 20″ and method 40, 40″ having the complementarymodified monomers M advantageously have more permutations of sequencesfor a given length of capture probe 2C and anti-capture region α2C. Forexample, in the preferred oligonucleotide capture probes 2C andanti-capture regions α2C of the solution probes 2S, the use of reversedpolarity nucleotides {overscore (N)} will allow more “letters” withwhich to make unique “words”; that is, there will be eight uniquenucleotide bases, instead of four with which to design probes 2C and 2S.Thus, there are the four nucleotides A, T (U), C, and G withnon-reversed polarity N and four nucleotides {overscore (A)}, {overscore(T)} ({overscore (U)}), {overscore (C)} and {overscore (G)} withreversed polarity {overscore (N)} with which to form unique sequences inthe capture probes 2C and anti-capture regions α2C of the solutionprobes 2S to generate unique probe sequences for an assay.

For example, if a probe is desired with a length of 20 nucleotides, thenusing the four non-reversed polarity nucleotide bases N, one can achieve4{circumflex over ( )}20 (=1.1 EA{circumflex over ( )}+12) combinationsof sequences. If these combinations are screened to remove thosecombinations that are likely to cross-hybridize using the methoddescribed by Brenner or others, referenced above, to provide sequenceswith minimal cross-hybridization, then a far lower number of usefulsequences is actually achieved. An even lower number would result with asecondary screening, using more rigorous criteria such as removingmismatch sequences that pass typical symbolic screenings but arefiltered out using thermodynamic parameters of mismatches, such as thatdescribed in U.S. Pat. No. 5,556,749 (Mitsuhashi et al.) issued Sep. 17,1996. An even lower number would result with a tertiary screening forintramolecular structures using conventional methods, such as thatdescribed by D. H. Mathews, T. C. Andre, J. Kim, D. H. Turner, and M.Zuker (1998) American Chemical Society Symposium Series 682, 246-257; N.B. Leontis and J. Santa Lucia Jr., eds., for example, and the referencescited therein, all of which are incorporated herein by reference.

In contrast, the addition of the four reversed polarity nucleotides{overscore (N)} with the four non-reversed polarity nucleotides N, asprovided in the system 20, 20″ and method 40, 40″ of the presentinvention, provides a total of eight unique nucleotides {overscore (N)},N. Each of the eight unique nucleotides {overscore (N)}, N can be usedin each sequence position of the capture probe 2C example above having20-nucleotides in length. Thus, there are 8{circumflex over ( )}20 (=1.2E{circumflex over ( )}+18) combinations of sequences available. Afterconventional screening of these combinations for cross-hybridization andintramolecular structures, as above, a much larger set remains thanwould be possible with just the four natural or non-reversed polaritynucleotides N. In fact, the thermodynamic specificity that thecomplementary reversed polarity nucleotides {overscore (N)} have foreach other systematically provides a reduced likelihood that crosshybridizations and intramolecular structures will occur even before thesets are screened. Therefore, the number of useful sequence combinationsfor the system 20, 20″ and method 40, 40″ of the present invention iseven larger and the length of the probes can be shorter. Shorter probelengths advantageously have lower synthesis costs and faster turn-aroundtime.

In fact, there are enough unique combinations of sequences using thereversed polarity nucleotides {overscore (N)} of the present inventionwith the non-reversed polarity nucleotides N that probe lengths L asshort as six nucleotides total comprising both reversed polaritynucleotides {overscore (N)} and non-reversed polarity nucleotides N(collectively “{overscore (N)}, N”) are possible. In a nucleotide probelength of L=6 nucleotides total example, one can achieve either4{circumflex over ( )}6 (=4,096) conventional, or 8{circumflex over ()}6 (=2.6 E{circumflex over ( )}+05) unique, combinations of sequencesusing either the 4 non-reversed N, or 8 reversed and non-reversed{overscore (N)}, N nucleotides, respectively. However, depending uponthe washing stringency, the shortest oligonucleotide probe or regionlength L which has good hybridization strength is preferably L=15 totalnucleotides {overscore (N)}, N for the system 20, 20″ that includes across-linking pair Y-Z, and preferably, L=25 total nucleotides{overscore (N)}, N for the system 20, 20″ without a cross-linking pairY-Z.

For the sandwich hybridization assays on arrays, especially multiplexingarray assays, as provided by the assay system 20″ and method 40″, thereare preferably over 10,000 different capture probe 2C features to besupported with the possible sequence combinations. It should be apparentthat the four non-reversed polarity nucleotide bases N provide a numberof combinations after screening for minimal cross-hybridization andintramolecular structures that may be too low to support an array ofover 10,000 features, depending upon the rigorousness of the screenings.To compensate for this inadequacy, the conventional systems would haveto use progressively longer probe sequences to provide enoughcombinations after screening to support a large array of over 10,000features. The longer the probe sequence, the more costly and lessdesirable the systems become.

Advantageously, a larger number of unique sequence combinations areavailable using the eight nucleotide bases {overscore (N)}, N, asprovided by the system 20, 20″ and method 40, 40″ of the presentinvention, before and after screening for minimal cross-hybridizationsand intramolecular structures. Therefore, the system 20, 20″ and method40, 40″ of the present invention provide for sets of much greater than10,000 capture probes 2C and anti-capture regions α2C for solutionprobes 2S that are unique with respect to each other and can be shorterthan the conventional probe sequences. Advantageously, these sets ofprobes and sequence regions 2C and α2C are suitable for supporting thesandwich hybridization assay system 10 and method 30 for multiplexingwell over 10,000 biological samples or biological targets on a singlearray. Thus, the capture probes 2C and complementary anti-capturesequences α2C of the system 20, 20″ and method 40, 40″ advantageouslyprovide for probes 2C and sequence regions α2C that support arrays ofover 10,000 features, that can be shorter in length, that have goodspecificity and sensitivity, and that are likely less costly to producethan the current state of the art. These capture probes 2C and solutionprobes 2S of system 20, 20″ and method 40, 40″ are readily adaptable foruse with the multiplexing system 10 and method 30 of the invention.

As mentioned above, an important advantage of both systems 10, 20, 20″and methods 30, 40, 40″ of the present invention is that customizationof a sandwich hybridization assay resides in the preparation of thesolution probes S, 2S, rather than in the preparation of the captureprobes C, 2C bound to the apparatus 11, 21, 21″. Therefore,manufacturing of the apparatus 11, 21, 21″ will be more cost-effectiveand quicker due to the common or universal pool or set of capture probesC, 2C for all target biological material T, 2T and all samples P. Thecurrent state-of-the-art requires custom probes on an assay substrate,including an array substrate. In contrast, the assay apparatus 21,including the array apparatus 11, 21″ can be fabricated in bulk andstored to allow cost savings compared to synthesizing custom is probeson the apparatus of the conventional systems. The bulk fabricatedapparatus 11, 21, 21″ will have the generic set of capture probes C, 2Ceither synthesized in situ, or pre-synthesized and deposited, on thesubstrate 12, 22, 22″. Moreover, the set of capture probes C, 2C of theinvention can be synthesized in bulk and stored to allow cost savingscompared to the cost of synthesizing different capture probes for eachdifferent type of custom assay. The pre-synthesized capture probes C, 2Ccan be spotted or chemically or enzymatically linked to the assaysubstrate 22 or to the appropriate feature location on the arraysubstrate 12, 22″ of the apparatus 11, 21, 21″. In either case, largenumbers of generic or universal assay apparatuses 21, including arrayapparatuses 11, 21″ can be manufactured at a time, since thecustomization is in the solution probes S, 2S, thereby saving in costand turnaround time for custom orders.

The kit, as mentioned above for system 10 and 30, can be provided withthe system 20, 20″ and written instructions for the method 40, 40″. Thekit provides sandwich hybridization assay capabilities to a user havinggood specificity and sensitivity, wherein such assay capabilitiesinclude multiplexing arrays, as provided by system 10 and 30.

The fabricated apparatus 11, 21, 21″ of the invention is used toevaluate polynucleotide or oligonucleotide “target” samples to betested. A user will expose the apparatus 11, 21, 21″ to one or moresamples, such as in hybridizing or binding assays, and interrogate thearray following such exposure using well-known conventional methods. Theinterrogation will produce a result. Information about the targetsample(s) can be obtained from the results of the interrogation. Theuser may be in a location remote to the location where the apparatus isfabricated. Moreover, the user may communicate the results or theinformation obtained from the results to a location remote to the user'slocation. A location is remote if it is at least a different location,e.g., a different building, a different city, different state ordifferent country, or if the location is at least one, at least ten, orat least one hundred miles apart.

Another advantage of the present systems 10, 20, 20″ and methods 30, 40,40″ of the invention is that the binding or hybridization between thetarget material T, 2T and the solution probe S, 2S (steps 36a, 46a)advantageously can occur in solution, as opposed to binding orhybridization on the surface 13, 23, 23″ of the apparatus 11, 21, 21″.Hybridization in solution advantageously allows for more effectivekinetics of binding or hybridization, and more control over thetemperature, mixing, and solution properties in the binding orhybridization step (36a, 46a). Since a particular target material T, 2Tcould be a large molecule, the anti-target sequence αT, α2T of thesolution probe S, 2S may have to be a large molecule also, such as acDNA, an antibody, etc., that is complementary to the large moleculetarget material T, 2T. A large anti-target sequence αT, α2T, having alarge target molecule T, 2T attached, advantageously can be indirectlylinked to the array apparatus 11, 21″ through addressable, self-assemblydescribed above, as opposed to current spotting techniques. Currentspotting techniques can be accompanied by irreversible denaturation. Theanti-target sequence αT, α2T of the solution probe S, 2S binds orhybridizes to the target T, 2T while in solution, so advancedinstrumentation is not needed for the physical placement of molecules bymicro-pipetting or masking of the array surface 13, 23″ for example. Inaddition, the activity of the large molecules is easier to maintain insolution.

Moreover, the manufacture of the assay apparatus 11, 21, 21″ used in thesystems 10, 20 and methods 30, 40 of the present invention usesconventional materials and processes. The substrates 12, 22, 22″ aremade of glass, fused silica or clear plastics, and preferably siliceousglass (i.e. silica-based glass) is used in the invention because of itslow intrinsic fluorescence. The siliceous glass can be obtained fromErie Scientific (Portsmouth, N.H.) or Coming (Coming, N.Y.). The captureprobes C, 2C are either synthesized in situ directly onto the substrate12, 22, 22″ or pre-synthesized and deposited onto the substrate 12, 22,22″ as intact species, using conventional methods. The monomers areadded to the substrate 12, 22, 22″ using the technology concepts fromthe thermal ink jet printing systems made by Hewlett-Packard of PaloAlto, Calif., or piezoelectric printing systems, made by Epson of Japan,for example. The array apparatuses 11, 21″ are manufactured usingautomated equipment, such that the spatial location on the substrate ofeach feature location is known within a certain margin of error.

Analysis (step 39, 49) of the assay is typically performed withcommercially available optical scanning systems, examples of which aredescribed in U.S. Pat. No. 5,837,475, U.S. Pat. No. 5,760,951 (confocalscanner) and U.S. Pat. No. 5,585,639 (off axis scanner), allincorporated herein by reference. Typical scanning fluorometers arecommercially available from different sources, such as MolecularDynamics of Sunnyvale, Calif., General Scanning of Watertown, Mass.,Hewlett Packard of Palo Alto, Calif. and Hitachi USA of So. SanFrancisco, Calif. Analysis of the data, (i.e., collection,reconstruction of image, comparison and interpretation of data) isperformed with associated computer systems and commercially availablesoftware, such as IMAGEQUANT™ by Molecular Dynamics. Moreover, thepresent invention does not require that optical interrogation be used toevaluate an assay. Advantageously, the present invention can use anyinterrogation or detection systems or methods.

Thus there has been described new systems, tools and methods of assayingbiological materials. The array assays of the invention areadvantageously addressable and self-assembling using the systems, toolsand methods of the invention. Multiplexing of one or a plurality of thesame or different samples and one or a plurality of the same ordifferent targets per sample are possible on the single array apparatuswith the systems, tools and methods of the invention. Good specificityand sensitivity of the systems and tools provide more accurate assayresults. The assay tools are relatively inexpensive to make since theinvention provides for generic or universal assay apparatuses withcapture probes and separately provides custom solution probes. It shouldbe understood that the above-described embodiments are merelyillustrative of the some of the many specific embodiments that representthe principles of the present invention. Clearly, numerous otherarrangements can be readily devised by those skilled in the art withoutdeparting from the scope of the present invention.

What is claimed is:
 1. An assay system for multiplexing together on asingle array a plurality of different biological samples having onebiological target per sample or a plurality of different biologicaltargets per sample, comprising: an array apparatus that comprises aplurality of first biological probes on a substrate in an array patternof features, wherein each first probe is different from others in theplurality of first probes, each different first probe is located at adifferent feature location of the array and is a different address onthe array; and a plurality of second biological probes, each secondprobe comprising a first region and a second region, each second probebeing different from others in the plurality of second probes bycomprising a different first region, wherein the first region of eachsecond probe is complementary to a different first probe, and the secondregion of each second probe is complementary to a specific target, suchthat each second probe addresses a complementary specific target from adifferent sample of the plurality of different samples being assayed toone of the different addresses on the array corresponding to arespective complementary different first probe.
 2. The assay system ofclaim 1, wherein the plurality of different biological samples comprisesthe one target per sample to be multiplexed, and wherein the secondregion of each second probe is the same and is complementary to the onetarget per sample in the plurality of different samples, such that thetarget from each different sample is distinguishable according to thecorresponding address of the respective first probe on the array.
 3. Theassay system of claim 1, wherein the plurality of different biologicalsamples comprises the plurality of different targets per sample to bemultiplexed, and wherein the second region of each second probe isdifferent and is complementary to a different one of the plurality oftargets per sample in the plurality of samples, such that each differenttarget and each different sample are distinguishable according to thecorresponding address of the respective first probe on the array.
 4. Theassay system of claim 1, wherein the plurality of first biologicalprobes comprises a group selected from oligonucleotides, antigens,antibodies, receptors or ligands.
 5. The assay system of claim 4,wherein the first regions of the plurality of second biological probescomprise a group complementary to the plurality of first probes selectedfrom oligonucleotides, antigens, antibodies, receptors or ligands. 6.The assay system of claim 1, wherein each first probe and eachcomplementary first region comprises a member of a specific binding pairthat cross-links to each other during the assay.
 7. The assay system ofclaim 1, wherein the second regions of the plurality of second probescomprise a group complementary to the one or more biological targets andis selected from oligonucleotides, cDNAs, PCR products, antigens,antibodies, receptors or ligands.
 8. The assay system of claim 1,wherein the plurality of first probes comprises multiple copies of eachfirst probe at each respective feature location, and wherein theplurality of second probes comprises multiple copies of each secondprobe.
 9. An assay system for multiplexing on a single array one or morebiological samples having one or more biological targets per sample,comprising: an array apparatus that comprises a plurality of firstbiological probes on a substrate in an array pattern of features,wherein each first probe is different from others in the plurality offirst probes, each different first probe is located at a differentfeature location of the array and is a different address on the array;and a plurality of second biological probes, each second probecomprising a first region and a second region, each second probe beingdifferent from others in the plurality of second probes by comprising adifferent first region, wherein the first region of each second probe iscomplementary to a different first probe, and the second region of eachsecond probe is complementary to a target in the one or more samples,such that each second probe addresses a target from a sample beingassayed to one of the different addresses on the array corresponding toa respective complementary different first probe, wherein each firstprobe comprises a first sequence of monomers, and wherein the firstregion of each second probe comprises a second sequence of monomers, andwherein the first sequence of monomers comprises a first modifiedmonomer and the second sequence of monomers comprises a second similarlymodified monomer that is complementary to the first modified monomer,wherein the modification causes the complementary first modified monomerand second modified monomer to preferentially hybridize or bind witheach other instead of with a complementary monomer that is not similarlymodified.
 10. An assay system for multiplexing on a single array one ormore biological samples having one or more biological targets persample, comprising: an array apparatus that comprises a plurality offirst biological probes on a substrate in an array pattern of features,wherein each first probe is different from others in the plurality offirst probes, each different first probe is located at a differentfeature location of the array and is a different address on the array;and a plurality of second biological probes, each second probecomprising a first region and a second region, each second probe beingdifferent from others in the plurality of second probes by comprising adifferent first region, wherein the first region of each second probe iscomplementary to a different first probe, and the second region of eachsecond probe is complementary to a target in the one or more samples,such that each second probe addresses a target from a sample beingassayed to one of the different addresses on the array corresponding toa respective complementary different first probe, wherein the pluralityof first probes comprises oligonucleotide first probes and the firstregions of the plurality of second probes comprises oligonucleotideregions, and wherein the oligonucleotide first probes comprise aplurality of first nucleotides having either a (3′→5′) or (5′→3′)polarity, and a first modified nucleotide that has a reversed polaritywith respect to the polarity of the plurality of first nucleotides, andwherein the oligonucleotide regions of the second probes comprise aplurality of second nucleotides having the same polarity as the polarityof the plurality of first nucleotides, the plurality of secondnucleotides being complementary to the plurality of first nucleotides,and a second modified nucleotide that has a reversed polarity withrespect to the polarity of the plurality of second nucleotides, thesecond reversed polarity nucleotide being complementary to the firstreversed polarity nucleotide in the first probe, and wherein thecomplementary first reversed polarity nucleotide and the second reversedpolarity nucleotide preferentially hybridize with each other instead ofwith a complementary nucleotide whose polarity is not similarlyreversed, such that specificity and sensitivity is provided to theassay.
 11. The assay system of claim 1, wherein the plurality of firstprobes is generic to the plurality of different biological samples andto the one target per sample or the plurality of different biologicaltargets per sample being assayed, and wherein the plurality of secondprobes is custom to the one target or the plurality of differentbiological targets being assayed and to the plurality of first probes.12. An assay method of multiplexing together on a single array aplurality of different biological samples having one biological targetper sample or a plurality of different biological targets per sample,comprising the steps of: providing an array apparatus having a pluralityof first biological probes on a substrate in an array pattern offeatures, each first probe being different, each different first probebeing located on a different feature location of the array, wherein eachdifferent first probe is a different address on the array apparatus;providing a plurality of second biological probes, each second probecomprising a first region and a second region, each second probe beingdifferent from others in the plurality of second probes by comprising adifferent first region that is complementary to a different one of thefirst probes, the second region of each second probe being complementaryto a specific target, such that each second probe addresses acomplementary specific target from a different sample of the pluralityof different samples being assayed to one of the different addresses onthe array corresponding to a respective complementary different firstprobe; assembling the target from each sample to the array with theplurality of second probes; removing unassembled biological materialsfrom the array; and analyzing assay results comprising the step ofdetermining the presence of the one target or the plurality of differenttargets in the plurality of different samples from whether the targetfrom each different sample is assembled on the array at thecorresponding first probe location.
 13. The assay method of claim 12,wherein the step of assembling comprises the steps of: incubating theplurality of different biological samples with the plurality of secondprobes to hybridize the biological targets per sample with complementarysecond regions of the second probes; and incubating together theplurality of second probes with the plurality of first probes on thearray apparatus to hybridize the first regions of the plurality ofsecond probes to corresponding complementary first probes.
 14. The assaymethod of claim 12, wherein the step of removing comprises the step ofwashing any unhybridized second probes and any unhybridized targets offthe array apparatus surface.
 15. The assay method of claim 12, whereinthe plurality of different biological samples comprises the one targetper sample to be multiplexed, wherein the second region of each secondprobe is the same and complementary to the one target per sample, andwherein the step of assembling comprises the steps of: separatelypreincubating a different second probe with each different sample of theplurality of different samples so that each different second probehybrdizes to the complementary target from a respective sample, andincubating together the different hybridized second probes with thearray apparatus to hybridize the different first regions of thedifferent hybridized second probes with complementary first probes onthe array corresponding to the different addresses.
 16. The assay methodof claim 12, wherein the plurality of different biological samplescomprises the plurality of different targets per sample to bemultiplexed, wherein the second region of each second probe is differentand complementary to a different one of the plurality of differenttargets per sample, and wherein the step of assembling comprises thesteps of: separately preincubating different second probes with eachdifferent sample, the different second probes in a respective samplebeing complementary to the different targets of the respective sample sothat each different second probe in the respective sample hybridizes tothe complementary target of the respective sample, and incubatingtogether the different hybridized second probes with the array apparatusto hybridize the different first regions of the different hybridizedsecond probes with complementary first probes on the array correspondingto the different addresses.
 17. An assay method of multiplexing on asingle array one or more biological samples having one or morebiological targets per sample, comprising the steps of: providing anarray apparatus having a plurality of first biological probes on asubstrate in an array pattern of features, each first probe beingdifferent, each different first probe being located on a differentfeature location of the array, wherein each different first probe is adifferent address on the array apparatus; providing a plurality ofsecond biological probes, each second probe comprising a first regionand a second region, each second probe being different from others inthe plurality of second probes by comprising a different first regionthat is complementary to a different one of the first probes, the secondregion of each second probe being complementary to a target from the oneor more samples, such that each second probe addresses a target from asample being assayed to one of the different addresses on the arraycorresponding to a respective complementary different first probe;assembling the target from the sample to the array with the plurality ofsecond probes; removing unassembled biological materials from the array;and analyzing assay results comprising the step of determining thepresence of the one or more targets in the one or more samples fromwhether the target from the sample is assembled on the array at thecorresponding first probe location, wherein each first probe comprises afirst sequence of monomers, and wherein the first region of each secondprobe comprises a second sequence of monomers, and wherein the firstsequence of monomers comprises a first modified monomer and the secondsequence of monomers comprises a second similarly modified monomer thatis complementary to the first modified monomer, and wherein in the stepof assembling, the first modified monomer preferentially hybridizes tothe complementary second modified monomer instead of to a complementarymonomer that is not similarly modified, such that the method providesspecificity and sensitivity to the assay.
 18. An assay method ofmultiplexing on a single array one or more biological samples having oneor more biological targets per sample, comprising the steps of:providing an array apparatus having a plurality of first biologicalprobes on a substrate in an array pattern of features, each first probebeing different, each different first probe being located on a differentfeature location of the array, wherein each different first probe is adifferent address on the array apparatus; providing a plurality ofsecond biological probes, each second probe comprising a first regionand a second region, each second probe being different from others inthe plurality of second probes by comprising a different first regionthat is complementary to a different one of the first probes, the secondregion of each second probe being complementary to a target from the oneor more samples, such that each second probe addresses a target from asample being assayed to one of the different addresses on the arraycorresponding to a respective complementary different first probe;assembling the target from the sample to the array with the plurality ofsecond probes; removing unassembled biological materials from the array;and analyzing assay results comprising the step of determining thepresence of the one or more targets in the one or more samples fromwhether the target from the sample is assembled on the array at thecorresponding first probe location, wherein the plurality of firstprobes comprises oligonucleotide first probes and the first regions ofthe plurality of second probes comprises oligonucleotide regions, andwherein the oligonucleotide first probes comprise a plurality of firstnucleotides having either a (3′→5′) or (5′→3′) polarity, and a firstmodified nucleotide that has a reversed polarity with respect to thepolarity of the plurality of first nucleotides, and wherein theoligonucleotide regions of the second probes comprise a plurality ofsecond nucleotides having the same polarity as the polarity of theplurality of first nucleotides, the plurality of second nucleotidesbeing complementary to the plurality of first nucleotides, and a secondmodified nucleotide that has a reversed polarity with respect to thepolarity of the plurality of second nucleotides, the second reversedpolarity nucleotide being complementary to the first reversed polaritynucleotide in the first probe, and wherein in the step of assembling,the plurality of first probes are hybridized to the plurality of secondprobes and the first reversed polarity nucleotide preferentiallyhybridizes to the complementary second reversed polarity nucleotideinstead of to a complementary nucleotide whose polarity is not similarlyreversed, such that the method provides specificity and sensitivity tothe assay.
 19. The assay method of claim 13, wherein each first probeand each complementary first region further comprises a member of aspecific binding pair, and wherein the step of assembling furthercomprises the step of cross-linking the members of the pair.
 20. Theassay method of claim 13, wherein the steps of incubating are performedsimultaneously.
 21. The assay method of claim 13, wherein the step ofincubating together the plurality of second probes with the plurality offirst probes is performed before the step of incubating the differentbiological samples with the plurality of second probes.
 22. The assaymethod of multiplexing of claim 12, wherein the assay results areanalyzed at a first location, the method further comprisingcommunicating the assay results or a conclusion based on the assayresults to a second location remote from the first location.
 23. Theassay method of multiplexing of claim 22, wherein the one or moresamples was obtained from a third location remote from the firstlocation or the second location.
 24. The assay system of claim 1, wherethe first regions of the plurality of second probes are hybridized tothe complementary first probes of the plurality of first probes.
 25. Theassay system of claim 1, wherein each first probe of the plurality offirst probes comprises a first sequence of monomers, and wherein thefirst region of each second probe of the plurality of second probescomprises a second sequence of monomers, and wherein the first sequenceof monomers comprises a first modified monomer and the second sequenceof monomers comprises a second similarly modified monomer that iscomplementary to the first modified monomer, wherein the modificationcauses the complementary first modified monomer and second modifiedmonomer to preferentially hybridize or bind with each other instead ofwith a complementary monomer that is not similarly modified.
 26. Theassay system of claim 1, wherein the plurality of first probes comprisesoligonucleotide first probes and the first regions of the plurality ofsecond probes comprises oligonucleotide regions, and wherein theoligonucleotide first probes comprise a plurality of first nucleotideshaving either a (3′→5′) or (5′→3′) polarity, and a first modifiednucleotide that has a reversed polarity with respect to the polarity ofthe plurality of first nucleotides, and wherein the oligonucleotideregions of the second probes comprise a plurality of second nucleotideshaving the same polarity as the polarity of the plurality of firstnucleotides, the plurality of second nucleotides being complementary tothe plurality of first nucleotides, and the second modified monomercomprises a second nucleotide that has a reversed polarity with respectto the polarity of the plurality of second nucleotides, the secondreversed polarity nucleotide being complementary to the first reversedpolarity nucleotide in the first probe, and wherein the complementaryfirst reversed polarity nucleotide and the second reversed polaritynucleotide preferentially hybridize with each other instead of with acomplementary nucleotide whose polarity is not similarly reversed, suchthat specificity and sensitivity is provided to the assay.
 27. The assaymethod of claim 12, wherein each first probe of the plurality of firstprobes comprises a first sequence of monomers, and wherein the firstregion of each second probe of the plurality of second probes comprisesa second sequence of monomers, and wherein the first sequence ofmonomers comprises a first modified monomer and the second sequence ofmonomers comprises a second similarly modified monomer that iscomplementary to the first modified monomer, and wherein in the step ofassembling, the first modified monomer preferentially hybridizes to thecomplementary second modified monomer instead of to a complementarymonomer that is not similarly modified, such that the method providesspecificity and sensitivity to the assay.
 28. The assay method of claim12, wherein the plurality of first probes comprises oligonucleotidefirst probes and the first regions of the plurality of second probescomprises oligonucleotide regions, wherein the oligonucleotide firstprobes comprise a plurality of first nucleotides having either a (3′→5′)or (5′→3′) polarity, and a first modified nucleotide that has a reversedpolarity with respect to the polarity of the plurality of firstnucleotides, and wherein the oligonucleotide regions of the secondprobes comprise a plurality of second nucleotides having the samepolarity as the polarity of the plurality of first nucleotides, theplurality of second nucleotides being complementary to the plurality offirst nucleotides, and a second modified nucleotide that has a reversedpolarity with respect to the polarity of the plurality of secondnucleotides, the second reversed polarity nucleotide being complementaryto the first reversed polarity nucleotide in the first probe, andwherein in the step of assembling, the plurality of first probes arehybridized to the plurality of second probes and the first reversedpolarity nucleotide preferentially hybridizes to the complementarysecond reversed polarity nucleotide instead of to a complementarynucleotide whose polarity is not similarly reversed, such that themethod provides specificity and sensitivity to the assay.